Recombinant dna comprising dna coding for myosin heavy chain sm1 iso-form protein inserted into vector dna microorganism carrying the recombinant dna, and an agent for treatment of arteriosclerosis comprising the recombinant dna

ABSTRACT

The present invention relates to recombinant DNA comprising DNA coding for smooth-muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA, a microorganism carrying the recombinant DNA, and an agent for treatment of arteriosclerosis comprising the recombinant DNA. The recombinant DNA of the present invention can be used effectively as an agent for gene therapy of restenosis after PTCA treatment.

TECHNICAL FIELD

The present invention relates to recombinant DNA comprising DNA coding for myosin heavy chain SM1 isoform protein inserted into vector DNA, a microorganism carrying the recombinant DNA and an agent for treatment of arteriosclerosis comprising the recombinant DNA which are used in gene therapy.

BACKGROUND ART

Smooth muscle-type myosin heavy chain SM1 isoform protein is responsible for contraction and relaxation of smooth muscles, and is one of the myosin heavy chain isoform proteins expressed specifically in smooth muscle cells. As the DNA coding for the protein, the nucleotide sequence of cDNA coding for rabbit SM1 isoform is known (P. Babij et al.: Proc. Natl. Acad. Sci. USA, 88, 10676 (1991)), but there is not known any homology among nucleotide sequences for such DNAs. Further, there is not known any recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA which recombinant DNA can be injected into animal cells.

It has been reported that when cDNA coding for a protein called calponin which is a protein existing in smooth muscle cells is introduced and expressed in a vascular smooth muscle cell line derived from rat pulmonary arteries, the time required for doubling the cells is prolonged by about 4 hours (Takahashi et al.: Circulation, 88, I-174 (1993)) and that when cDNA for human calponin is injected into a rabbit topically at a site where the carotid artery has been abraded with a balloon, thickening of the intima is inhibited (Takahashi et al.: Circulation, 88, I-656 (1993)). It is not known that recombinant DNA comprising DNA coding for myosin heavy chain SM1 isoform protein inserted into vector DNA has pharmacological effect and is used for in gene therapy.

For treatment of arteriosclerosis, percutaneous transluminal coronary angioplasty (PTCA) in which a stenosed site is enlarged by a balloon catheter is extensively conducted. The treatment method has the advantages of easier operation and higher degree of success than the bypass surgery etc., while the treatment method has the disadvantage that 30 to 40% of the patients after the treatment suffer from restenosis of blood vessels due to abnormal proliferation of vascular cells. Therefore, there is demand for developments in an agent for treatment of arteriosclerosis effectively used for preventing such restenosis.

DISCLOSURE OF THE INVENTION

The present invention includes:

(1) A recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA.

(2) The recombinant DNA according to (1) wherein the vector DNA is a retrovirus vector, adenovirus vector, adeno-associated virus vector or a plasmid capable of being expressed in an animal.

(3) The recombinant DNA according to (2) wherein the plasmid capable of being expressed in an animal is pCXN2 or PAGE208.

(4) The recombinant DNA according to any one of (1) to (3) wherein the smooth muscle-type myosin heavy chain SM1 isoform protein is of a human smooth muscle type, rabbit muscle type or mouse muscle type.

(5) The recombinant DNA according to any one of (1) to (3) wherein the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein is the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence shown in SEQ ID NO:1 in which at least one nucleotide is added, deleted or replaced.

(6) DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein, which is the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence shown in SEQ ID NO:1 in which at least one nucleotide is added, deleted or replaced.

(7) A microorganism carrying the recombinant DNA of (5).

(8) The microorganism according to (7) which belongs to the genus Escherichia.

(9) An agent for treatment of arteriosclerosis which comprises the recombinant DNA of any one of (1) to (5).

The DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein includes cDNA or genomic DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein, preferably DNA derived from humans, rabbits, mice, etc.

The nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein includes the nucleotide sequence of cDNA (SEQ ID NO:5) coding for rabbit SM1 isoform protein (SEQ ID NO:4) (Proc. Natl. Acad. Sci. USA, 88, 10676 (1991)), the nucleotide sequence of the DNA coding for mouse myosin heavy chain SM1 isoform protein as shown in SEQ ID NO:1, and the nucleotide sequence as shown in SEQ ID NO:2 which is specified by homologous regions between the above nucleotide sequences (In the Sequence Listing, Y represents T, U or C; S represents G or C; V represents A, G or C; B represents B, C, T or U; W represents A, T, or U; N represents A, C, G, T, U or a single bond; and the codon YAS (388-390) represents TAC or CAG.). Accordingly, the nucleotide sequence shown in SEQ ID NO:2 contains the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of the cDNA coding for rabbit SM1 isoform protein. A partial nucleotide sequence of cDNA coding for human smooth muscle-type myosin heavy chain SM1 isoform protein is also known (Amer. J. of Medical Genetics, 46, 61-67 (1993)), and a nucleotide sequence of this partial nucleotide sequence combined with a partial nucleotide sequence of SEQ ID NO:2 is also contained in the nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein according to the present invention.

Further, the nucleotide sequence of SEQ ID NO:2 or the combination of a partial nucleotide sequence of SEQ ID NO:2 and the nucleotide sequence of the DNA derived from human smooth muscles, in which one or more nucleotides have been added, deleted or replaced by means of site-directed mutagenesis (Nucleic Acid Research, 10, 6487-6508 (1982)), is also contained in the nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein according to the present invention.

The vector DNA that can be used includes virus vectors such as retrovirus vector, adenovirus vector and adeno-associated virus vector and plasmids capable of being expressed in an animal such as pCXN2 (Gene, 108, 193-200 (1991)) and PAGE207 (Japanese Patent Laid-Open Publication No. 46841/1994) as well as their modified vectors.

The agent for treatment of arteriosclerosis according to the present invention can be produced by compounding the recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA as an active ingredient together with a base used in agents for gene therapy. Just before administration, the pharmaceutical preparation can be used in gene therapy for arteriosclerosis, if necessary after encapsulation in liposomes etc. (Proc. Natl. Acad. Sci. USA., 90, 11307 (1993)).

Where the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein is inserted into a virus vector, a therapeutic agent can be produced by preparing virus particles containing the recombinant DNA and then compounding them together with a base used in agents for gene therapy (Nature Genet., 8, 42(1994)).

The base used in agents for gene therapy may be any base generally used in injections. The base includes, for example, distilled water, a salt solution of sodium chloride, a mixture of sodium chloride and an inorganic salt, or the like, solutions of mannitol, lactose, dextran, glucose, etc., solutions of amino acid such as glycine, arginine, etc., a mixed solution of an organic acid solution or a salt solution and glucose solution, and the like. Further, injections may be prepared in a usual manner as a solution, suspension or dispersion by adding adjuvant such as an osmotic pressure controlling agent, pH adjusting agent, vegetable oils such as sesame oil, soybean oil, etc. or surface active agents such as lecithin, non-ionic surface active agent, etc. to the above base. These injections can also be powdered, lyophilized, etc. to be dissolved just before use.

The agent for treatment of arteriosclerosis can be used as such in the case of a solution, and in the case of a solid it is dissolved in the base previously sterilized if necessary just before use in gene therapy.

The method for administration of the agent for treatment of arteriosclerosis according to the present invention involves topically administrating it into a patients who underwent the PTCA treatment, at a dose of 1 ng to 1 g per day or once after the surgery by means of a catheter etc. such that it can be absorbed into their vascular smooth muscle cells at the target site.

The agent for treatment of arteriosclerosis according to the present invention is safe in this dose range.

Hereinafter, the process for producing the recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA is described.

The recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA can be obtained according to the usual genetic engineering method described below or its modified method.

A DNA clone coding for smooth muscle-type myosin heavy chain SM1 isoform protein is detected in a cDNA library from tissues composed mainly of smooth muscles in the uterus, aorta, etc., according to the method described by A. Abe et al. (ECL direct DNA labeling detection system manual (Amersham)).

The recombinant DNA comprising the inserted DNA coding for myosin heavy chain SM1 isoform protein can be produced by ligating said DNA fragment to a downstream region from a promoter in a suitable vector DNA (J. Sambrook et al., Molecular Cloning, 2nd Ed., Vol. 1, Cold Spring Harbor Laboratory Press (1989)). Animal cells are used as a host, and a promoter derived from SV40, a promoter from retrovirus, a metallothionein promoter, β-actin promoter etc. can be utilized as the promoter. For the expression, the use of an enhancer is also effective.

The reaction conditions for the above-mentioned recombinant techniques are as follows: The digestion of DNA with a restriction enzyme is carried out by allowing the restriction enzyme in an amount of 0.1 to 100 units, preferably 1 to 3 units per μg DNA to act on 0.1 to 20 μg DNA in a reaction solution usually containing 2 to 200 mM preferably 10 to 40 mM Tris-HCl buffer, pH 6.0 to 9.5, preferably pH 7.0 to 8.0, 0 to 200 mM sodium chloride, and 2 to 20 mM preferably 5 to 10 mM magnesium chloride, at 20 to 70° C. (the optimum temperature is varied depending on the restriction enzyme used) for 15 minutes to 24 hours. The termination of the reaction can be effected usually by heating the reaction solution at 55 to 75° C. for 5 to 30 minutes or by inactivating the restriction enzyme with reagents such as phenol etc. The DNA fragment, or the gapped duplex DNA, generated by digestion with the restriction enzyme can be purified using Prep-A-Gene Matrix (Bio-Rad). The ligation of the DNA fragment can be effected using a DNA ligation kit (Takara Shuzo Co., Ltd.).

The recombinant DNA containing DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein thus constructed in this manner is used to produce a transformant.

The host for the plasmid capable of being expressed in an animal includes, for example, microorganisms belonging to the genus Escherichia, such as Escherichia coli K12·HB101 (H. W. Boyer et al.: J. Mol. Biol.,41, 459 (1969)), DH5 α (D. Hanahan: J. Mol. Biol., 166, 557 (1983)) etc. The transformation of microorganisms of the genus Escherichia can be effected according to the method of Cohen et al. (S. N. Cohen et al.: Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)).

The host used for the virus vector includes animal cells having the ability to produce viruses, such as monkey cell COS-7, Chinese hamster cell CHO, mouse cell BALB/3T3, human cell HeLa, etc.; the host for the retrovirus vector includes ΨCRE, ΨCRIP (Proc. Natl. Acad. Sci. USA, 85, 6460 (1988)), MLV (J. Virol., 65, 1202 (1991)) etc.; and the host for the adenovirus vector and adeno-associated virus vector includes 293 cells derived from human fetal kidney (“jikken Igaku” (Experimental Medicine), 12, 316 (1994)) etc. The introduction of the virus vector into animal cells can be effected using the calcium phosphate method (Virology, 52, 456 (1973)) etc.

The resulting transformant can be cultured in the following manner depending on the difference in the cell species to produce the recombinant DNA.

To culture the transformant from a microorganism of the genus Escherichia as the host, the suitable medium is a liquid medium containing a carbon source, nitrogen source, inorganic substance, etc. necessary for the growth of the transformant. The carbon source includes e.g. glucose, dextrin, soluble starch, sucrose, glycerol, etc. ; the nitrogen source includes e.g. an ammonium salt, peptone, casein, etc.; and the inorganic substance includes e.g. calcium chloride, sodium dihydrogenphosphate, magnesium chloride, etc. A yeast extract, vitamins, etc. may further be added. The pH of the medium is preferably about 5 to 8. To culture the microorganism of the genus Escherichia, the medium is preferably Terrific broth (K. D. Tartof et al.: Bethesda Res. Lab. Focus, 9, 12 (1987)) or the like. The transformant is cultured usually at about 15 to 43° C. for about 8 to 24 hours, if necessary under aeration or stirring. After the culture is finished, the recombinant DNA containing DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein can be obtained through purification by the Birnboim method (Nucleic Acid Res., 7, 1513 (1979)), etc.

To culture the transformant from animal cells as the host, the medium used includes a medium containing about 5 to 20% fetal bovine serum, such as 199 medium (Morgan et al.: Proc. Soci. Biol. Med., 73, 1 (1950)), MEM medium (H. Eagle: Science, 122, 501 (1952)), DMEM (R. Dulbecco et al.: Virology, 8, 396 (1959)) or the like. The pH is preferably in the range of about 6 to 8. The transformant is cultured usually at about 30 to 40° C. for about 18 to 60 hours, if necessary under aeration or stirring.

Because virus particles containing the recombinant DNA are released into the culture supernatant, the recombinant DNA containing DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein can be obtained from the supernatant by concentrating and purifying virus particles by the cesium chloride centrifugation method (Koji Sawada et al.: “Shin Seikagaku Jikken Kouza 2-V” (New Biochemistry Experimental Course 2-V), 33 (1992)), the polyethylene glycol precipitation method (Arch. Virol., 71, 185 (1982)), the filter concentration method (J. Cell. Biol., 111, 217 (1990)), etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction enzyme map of the cDNA coding for mouse smooth muscle-type myosin heavy chain SM1 isoform protein and the location of the coding region.

Plasmid name: SM-1 F text

Plasmid size: 6175 bp

Region in the frame: coding region

A: ApaI

B: BamHI

Sa: SacI

Sm: SmaI

FIG. 2 shows the area of CHO cell colonies selected with hygromycin after introduction of plasmid pSE-SM1-Hyg vs. the area of CHO cell colonies after introduction of plasmid PAGE208.

FIG. 3 shows the area of HeLa cell colonies selected with hygromycin after introduction of plasmid pSE-SM1-Hyg vs. the area of HeLa cell colonies after introduction of plasmid PAGE208.

FIG. 4 shows the comparison among the proliferation rates of the wild type, the PAGE208-introduced clone and the SM1 isoform-expressing clones (SM1-5-2-1, SM1-5-3-3) of CHO cells by the MTT method.

—◯— CHO

—Δ— PAGE208-1

—— SM1 5-2-1

—▪— SM1 5-3-3

FIG. 5 shows the degrees of thickening of the intima after introduction of the SM1 gene (Example) and β-galactosidase gene (Comparative Example) into the vessel walls in a balloon injured rabbit model.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in more detail with reference to Examples which however are not intended to limit the scope of the present invention.

EXAMPLE 1 Recombinant DNA Comprising DNA Coding for Mouse Smooth Muscle-type Myosin Heavy Chain SM1 Isoform Protein Inserted into Vector DNA

(a) Preparation of a Probe

Plasmids pSMHC29 and pIH61 in which a cDNA fragment coding for rabbit smooth muscle-type myosin heavy chain SM2 isoform protein had been cloned (J. Biol. Chem., 264, 9734-9737 (1989)) were used. These were digested with EcoRI (a product of Takara Shuzo Co., Ltd.; the restriction enzymes used hereinafter are products of Takara Shuzo Co., Ltd. unless otherwise specified) to prepare a cDNA fragment of rabbit smooth muscle-type myosin heavy chain SM2 isoform protein to be inserted. The cDNA fragment was labeled using an ECL direct DNA labeling system (Amersham) to be used as a probe in the following screening.

(b) Screening

A λ gtll vector mouse uterus cDNA library (Clontech) was mixed with E. coli Y1090 (Clontech) and incubated at 37° C. for 15 minutes, followed by adding 0.7% agarose NZY (Nucleic Acid Res., 16, 7583-7600 (1988)), and it was then spread on a 1.5% agar NZY plate. A nylon filter was placed on the plate wherein plaques occurred so that the plaques were transferred to the filter. This filter was subjected to denaturation by alkali treatment and the DNA was fixed by heating at 80° C. for 2 hours. The DNA was hybridized to the probe previously labeled according to the protocol of the ECL direct DNA labeling system in (a) above. Then, the hybridized clones were detected by autoradiography using an ECL detection system (Amersham).

(c) Analysis of DNA Nucleotide Sequence

Four clones were identified and each clone was subcloned in pUC119 (Takara Shuzo Co., Ltd.) or pBluescript SK(−) (Stratagene) for analysis of the DNA nucleotide sequence. E. coli DH5α was transformed with the resulting plasmids to give transformants Escherichia coli DH5α/pmsmhc20, pmsmhcC5, pmsmhcN08 and pmsmhcN14. The plasmids produced by the transformants were subjected to stepwise deletion by digestion with an exonuclease or to digestion with a suitable restriction enzyme, followed by self-cyclization or subcloning to prepare template DNA for sequence analysis (Gene, 28, 351-359 (1984)). A fluorescence type DNA sequencer (Applied Biosystems) was used for sequencing and MacMolly (Soft Gene GmbH) was used for data analysis. The nucleotide sequence thus determined is shown in SEQ ID NO:1. The amino acid sequence deduced from the nucleotide sequence is also shown in SEQ ID NO:1.

(d) Construction of an Expression Plasmid for Mouse Smooth Muscle-type Myosin Heavy Chain SM1 Isoform

From the 4 clones previously obtained, plasmid pmSM1 containing cDNA coding for the whole coding region for SM1 isoform was prepared in the following manner (J. Sambrook et al., Molecular Cloning, 2nd Ed., Vol. 1, Cold Spring Harbor Laboratory Press, 1989). To shorten the 5′-non-coding region, plasmid pmsmhcN14 i.e. one of the 4 clones was digested with restriction enzymes BamHI and BglII and a 5-kb DNA fragment was purified and recovered therefrom. The DNA fragment was self-cyclized and transformed into E. coli DH5α to give an ampicillin resistant colony. From the colony, a desired microorganism is picked up and cultured, and plasmid DNA was recovered using a known method (H. C. Birnboim et al.: Nucleic Acid Res., 7, 1513 (1979)). The structure of the plasmid thus obtained was confirmed by agarose gel electrophoresis after digestion with BamHI and KpnI. The plasmid was designated pmsmhcN14′.

The resulting plasmid pmsmhcN14′ was digested with restriction enzymes NsiI (New England Biolabs) and EcoRV, and a 4-kb DNA fragment was purified and recovered therefrom. Separately, plasmid pmsmhcN08 was also digested with NsiI and EcoRV, and a 1-kb DNA fragment was purified, recovered and ligated into the previously obtained 4-kb DNA fragment derived from plasmid pmsmhcN14′, whereby plasmid pmsmhcN14-08 was constructed. The structure of this plasmid was confirmed by digestion with NsiI and EcoRV. The resulting plasmid pmsmhcN14-08 was digested with restriction enzymes ApaI and KpnI and a 5-kb DNA fragment was purified and recovered therefrom. Plasmid pmsmhc20 was also digested in a similar manner with Apal and KpnI, and a 1.5-kb DNA fragment was purified, recovered and ligated into the previously obtained 5-kb DNA fragment, whereby plasmid pmsmhcNl4-08-20 was constructed. The structure of the plasmid was confirmed by digestion with ApaI and KpnI.

The resulting plasmid pmsmhcN14-08-20 was digested with restriction enzyme ApaI and a 6-kb DNA fragment was purified and recovered therefrom. Plasmid pmsmhcC5 was also digested with ApaI, and a 2-kb DNA fragment was purified, recovered and ligated into the 6-kb DNA fragment from pmsmhcN14-08-20 whereby plasmid pmsmhcN14-08-5-20 was constructed. The structure of this plasmid was confirmed by digestion with EcORI and NruI.

Plasmid pmsmhcN14-08-5-20 was digested with restriction enzymes NsiI and NruI and a 8-kb DNA fragment was purified and recovered therefrom. Plasmid pmsmhcN08 was also digested in a similar manner with NsiI and NruI, and a 1.5-kb DNA fragment was purified, recovered and ligated into the previously obtained 8-kb DNA fragment, whereby plasmid pmSM1 containing the whole coding region for mouse smooth muscle-type myosin heavy chain SM1 isoform was constructed. The structure of this plasmid was confirmed by digestion with NsiI and NruI.

The resulting plasmid pmSM1 was digested with restriction enzymes XbaI and KpnI and a 6-kb DNA fragment was purified and recovered therefrom. Separately, expression vector PAGE208 derived from expression vector PAGE207 (Japanese Patent Laid-Open Publication No. 46841/1994) by deleting an SmaI site located in a promoter region for hygromycin B resistance gene in PAGE207 was digested with XbaI and KpnI, and a 6-kb DNA fragment was purified, recovered, and ligated into the previously obtained 6-kb DNA fragment derived from plasmid pmSM1, whereby expression plasmid pSE-SM1-Hyg was constructed. The structure of the plasmid was confirmed by digestion with XbaI and KpnI.

A DNA fragment obtained by digestion of plasmid pmSM1 with XbaI and KpnI was blunt-ended with a DNA blunting kit (Takara Shuzo Co., Ltd.). Separately, expression vector pCXN2 was digested with XhoI and blunt-ended with the DNA blunting kit (Takara Shuzo Co., Ltd.). These 2 kinds of DNA were ligated to construct expression plasmid pCAG-SM1. The structure of the plasmid was confirmed by digestion with BglII.

The above plasmid pSE-SM1-Hyg was inserted into Escherichia coli whereby a transformant was obtained.

The transformant has been deposited as Escherichia coli pSE-SM1-Hyg (FERM BP-4974) with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, Japan, since Jan. 24, 1995.

EXAMPLE 2 Effect of Expression of SM1 Isoform in CHO Cells and HeLa Cells on Cell Proliferation

2 μg of expression plasmid pSE-SM1-Hyg and 4 μl of lipofectamine™ (Gibco) were mixed and incubated for 15 minutes to produce a complex. The complex was added to CHO cells or HeLa cells and incubated at 37° C. for 5 hours so that the gene was introduced into the cells. The cells were diluted and spread on a vessel, and cells into which the gene had been introduced were selected in the presence of hygromycine™ (Sigma). About 10 days later, the drug resistant cell colonies were stained with Coomassie Blue™ (Bio-Rad) and their area was measured with an image analyzer. As a result, many of the colonies had smaller areas than those of the colonies carrying the introduced control vector PAGE208, indicating that their proliferation was inhibited by the expression of the SM1 isoform (FIGS. 2 and 3). Separately, the hygromycin resistant CHO cells were cloned and the resulting clones SM1-5-2-1 and SM1-5-3-3 were examined for their proliferation rate. The result indicated that their proliferation rate was lower than that of the wild-type or PAGE208-introduced cells (FIG. 4).

EXAMPLE 3 Inhibitory Effect of Forcible Introduction of SM1 Gene into Vessel Walls in a Balloon Injured Rabbit Model on Thickening of the Intima

A PTCA catheter at 3 French (Fr) was inserted under X-raying through the femoral artery into the right common carotid artery of a Japanese white domestic rabbit weighing 2.4 to 3.2 kg. The right common carotid artery was abraded 3 times with a balloon expanded at 10 atm. to injure the vessel wall. Three days after the abrasion, the injured portion of the vessel was infused with a mixture of 300 mg pCAG-SM1 and lipofectin (Gibco BRL) through a Wolinsky type infusion catheter (a product of BARD) at 6 atm. For Comparative Example, other domestic rabbits were treated in the same manner, and in place of pCAG-SM1, an expression vector for β-galactosidase was introduced into the injured portion of the vessel.

Three days after the infusion, the animals were sacrificed by perfusion with heparin physiological saline and their right common carotid arteries were removed and cut thin, followed by adding 1 ml ISOGEN (Wako Pure Chemical Industries, Ltd.), and the tissues were homogenized with a Polytron homogenizer. The whole RNA was extracted by the AGPC method (P. Chomczynski and N. Sacchi: Anal. Biochem., 162, 156-159 (1987)), and the MRNA for SM1 was amplified by the RT-PCR method. The amplified DNAs from the Example (introduction of SM1 gene) and Comparative Example (introduction of β-galactosidase gene) and their fragments digested with restriction enzyme NheI were subjected to agarose gel electrophoresis. The SM1 band was detected strongly in the Example and faintly in the Comparative Example, and the band in only the Example was digested with NheI. The supported that the SM1 gene derived from the mouse was expressed in the Example because the digestion site of the restriction enzyme is not present in the DNA for SM1 derived from the domestic rabbit but present in the mouse SM1.

Two weeks after introduction of the gene, the rabbits were sacrificed by perfusion with PBS containing 2% paraformaldehyde and fixed, and their right common carotid arteries were removed. The part into which the infusion catheter had been introduced was dissected out and embedded in paraffin, and a thin round section of the vessel was prepared. One vessel was divided equally into 4 tissues and 1 section was prepared from each tissue and stained with hematoxylineosin. The vessel was divided along the internal elastic plate as the boundary into the intima and media, and the area of each was determined. The intima area/media area ratio was determined for each section and the mean was determined. The degree of thickening of the intima was determined using the mean. From the degree of thickening of the intima as determined for 3 animals in the Example and 3 animals in the Comparative Example, it was confirmed that thickening of the intima was inhibited by forcible introduction of the SM1 gene (FIG. 5).

The results suggested that plasmids pSE-SM1-Hyg and pCAG-SM1 possess the effect of depressing cell proliferation and are effective in restenosis of arteriosclerosis, caused by abnormal proliferation of vascular cells.

INDUSTRIAL APPLICABILITY

It was revealed that the proliferation of animal cells is inhibited when the recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA according to the present invention is expressed in the animal cells. The effect was also observed in human-derived HeLa cells. Therefore, the recombinant DNA of the present invention can be used effectively as an agent for gene therapy of restenosis after PTCA treatment.

5 1 6175 DNA Mus musculus CDS (105)...(6020) 1 agatttggac catcccagcc tgggatcagt gccagatccg agctctccat ccggtgtcct 60 cctgctaatc cacccccgga gtagatctgg gaccaccaga catc atg gcg cag aaa 116 Met Ala Gln Lys 1 ggg cag ctc agc gat gat gag aag ttc ctc ttt gtg gat aaa aac ttc 164 Gly Gln Leu Ser Asp Asp Glu Lys Phe Leu Phe Val Asp Lys Asn Phe 5 10 15 20 atg aac agc cca atg gct cag gcc gac tgg gta gcc aag aag ctg gtg 212 Met Asn Ser Pro Met Ala Gln Ala Asp Trp Val Ala Lys Lys Leu Val 25 30 35 tgg gtc cct tca gag aag cag ggc ttc gaa gca gcc agc atc aag gag 260 Trp Val Pro Ser Glu Lys Gln Gly Phe Glu Ala Ala Ser Ile Lys Glu 40 45 50 gag aag ggc gat gag gtg gtc gtg gag ttg gtg gaa aat gga aag aag 308 Glu Lys Gly Asp Glu Val Val Val Glu Leu Val Glu Asn Gly Lys Lys 55 60 65 gtc aca gtt ggc aaa gat gac atc caa aaa atg aac cca ccc aag ttc 356 Val Thr Val Gly Lys Asp Asp Ile Gln Lys Met Asn Pro Pro Lys Phe 70 75 80 tct aag gtg gag gac atg gca gag ctg acg tgc ctc aat gag gcc tct 404 Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu Asn Glu Ala Ser 85 90 95 100 gtg ctg cac aac ctg agg gag cga tac ttc tca ggc ctc atc tat acc 452 Val Leu His Asn Leu Arg Glu Arg Tyr Phe Ser Gly Leu Ile Tyr Thr 105 110 115 tac tct ggc ctc ttc tgt gtg gtg gtc aac ccc tac aag tac cta ccc 500 Tyr Ser Gly Leu Phe Cys Val Val Val Asn Pro Tyr Lys Tyr Leu Pro 120 125 130 atc tac tca gaa aag atc gtg gat atg tac aag ggc aag aag agg cat 548 Ile Tyr Ser Glu Lys Ile Val Asp Met Tyr Lys Gly Lys Lys Arg His 135 140 145 gag atg ccg cct cac atc tat gcc att gcc gac aca gcc tac aga agc 596 Glu Met Pro Pro His Ile Tyr Ala Ile Ala Asp Thr Ala Tyr Arg Ser 150 155 160 atg cta caa gat cgt gaa gac cag tcc att ctg tgc aca ggt gag tct 644 Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys Thr Gly Glu Ser 165 170 175 180 gga gcc gga aag aca gag aac aca cag aaa gtc ata cag tac ttg gct 692 Gly Ala Gly Lys Thr Glu Asn Thr Gln Lys Val Ile Gln Tyr Leu Ala 185 190 195 gtg gtg gcg tcc tcc cac aag ggc aag aaa gac agc agc atc acg ggg 740 Val Val Ala Ser Ser His Lys Gly Lys Lys Asp Ser Ser Ile Thr Gly 200 205 210 gag ctg gaa aag cag ctt cta cag gca aac cca atc ctg gag gct ttc 788 Glu Leu Glu Lys Gln Leu Leu Gln Ala Asn Pro Ile Leu Glu Ala Phe 215 220 225 ggc aat gcg aaa acc gtc aag aac gac aac tcc tct cgc ttt ggc aag 836 Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser Arg Phe Gly Lys 230 235 240 ttc att cgc atc aac ttc gat gtc act ggt tac att gta ggt gcc aat 884 Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile Val Gly Ala Asn 245 250 255 260 att gaa aca tat ctt ctg gaa aag tct agg gct att cga cag gct agg 932 Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Ile Arg Gln Ala Arg 265 270 275 gat gag aga aca ttt cac atc ttc tac tac ctg ctc gcc gga gcc aag 980 Asp Glu Arg Thr Phe His Ile Phe Tyr Tyr Leu Leu Ala Gly Ala Lys 280 285 290 gaa aag atg aaa agt gac ctg ctt ttg gag agc ttc aac agc tac aca 1028 Glu Lys Met Lys Ser Asp Leu Leu Leu Glu Ser Phe Asn Ser Tyr Thr 295 300 305 ttt tta tcc aat ggc ttt gtg ccc atc cca gct gca caa gat gat gag 1076 Phe Leu Ser Asn Gly Phe Val Pro Ile Pro Ala Ala Gln Asp Asp Glu 310 315 320 atg ttc cag gag aca ctg gaa gcc atg tct atc atg ggc ttc aat gaa 1124 Met Phe Gln Glu Thr Leu Glu Ala Met Ser Ile Met Gly Phe Asn Glu 325 330 335 340 gag gaa cag cta gcc atc ttg aag gta gta tca tct gtc ctt cag ctt 1172 Glu Glu Gln Leu Ala Ile Leu Lys Val Val Ser Ser Val Leu Gln Leu 345 350 355 gga aac att gtc ttc aag aag gag cga aac aca gac cag gca tcc atg 1220 Gly Asn Ile Val Phe Lys Lys Glu Arg Asn Thr Asp Gln Ala Ser Met 360 365 370 cct gat aac aca gcg gct cag aaa gtt tgc cac ctc gtg ggg att aat 1268 Pro Asp Asn Thr Ala Ala Gln Lys Val Cys His Leu Val Gly Ile Asn 375 380 385 gtg aca gat ttc act aga gcc atc ctg acc cca cgt atc aaa gtt gga 1316 Val Thr Asp Phe Thr Arg Ala Ile Leu Thr Pro Arg Ile Lys Val Gly 390 395 400 cgg gat gtg gtg cag aag gct cag acc aaa gaa cag gct gac ttc gcc 1364 Arg Asp Val Val Gln Lys Ala Gln Thr Lys Glu Gln Ala Asp Phe Ala 405 410 415 420 atc gag gcc tta gcc aag gcc acc tat gag cgc ctt ttc cga tgg att 1412 Ile Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu Phe Arg Trp Ile 425 430 435 ctc agc cgt gta aac aag gcc ttg gac aag acc cat cgg cag ggg gcc 1460 Leu Ser Arg Val Asn Lys Ala Leu Asp Lys Thr His Arg Gln Gly Ala 440 445 450 tcc ttc ctg ggc att ctg gat att gct ggg ttt gaa atc ttt gag gta 1508 Ser Phe Leu Gly Ile Leu Asp Ile Ala Gly Phe Glu Ile Phe Glu Val 455 460 465 aac tcc ttc gag cag ctg tgc atc aac tac acc aac gag aag ctg cag 1556 Asn Ser Phe Glu Gln Leu Cys Ile Asn Tyr Thr Asn Glu Lys Leu Gln 470 475 480 cag ctg ttc aac cac acg atg ttc atc ctg gag cag gaa gag tac cag 1604 Gln Leu Phe Asn His Thr Met Phe Ile Leu Glu Gln Glu Glu Tyr Gln 485 490 495 500 cga gag ggc atc gag tgg aac ttc atc gac ttc ggc ctg gac ctg cag 1652 Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly Leu Asp Leu Gln 505 510 515 cct agt att gag ctg att gag cgg ccg aac aac cct ccc ggt gtg ctg 1700 Pro Ser Ile Glu Leu Ile Glu Arg Pro Asn Asn Pro Pro Gly Val Leu 520 525 530 gcc ctg ctg gat gaa gaa tgc tgg ttc ccc aaa gct aca gac aag tct 1748 Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala Thr Asp Lys Ser 535 540 545 ttt gtg gag aag cta tgc tca gag cag gga aat cac ccc aaa ttt cag 1796 Phe Val Glu Lys Leu Cys Ser Glu Gln Gly Asn His Pro Lys Phe Gln 550 555 560 aag ccc aag cag cta aag gac aaa aca gag ttc tcc atc atc cac tat 1844 Lys Pro Lys Gln Leu Lys Asp Lys Thr Glu Phe Ser Ile Ile His Tyr 565 570 575 580 gct ggg aag gtg gac tac aat gca agt gcc tgg ctg acc aag aac atg 1892 Ala Gly Lys Val Asp Tyr Asn Ala Ser Ala Trp Leu Thr Lys Asn Met 585 590 595 gac ccg cta aat gac aat gtg aca tca ctc ctc aat gcc tcc tct gac 1940 Asp Pro Leu Asn Asp Asn Val Thr Ser Leu Leu Asn Ala Ser Ser Asp 600 605 610 aag ttt gtg gct gac ctg tgg aag gat gtg gac cgc att gtg ggg ctg 1988 Lys Phe Val Ala Asp Leu Trp Lys Asp Val Asp Arg Ile Val Gly Leu 615 620 625 gac cag atg gcc aag atg act gag agc tca ctg ccc agt gcc tca aag 2036 Asp Gln Met Ala Lys Met Thr Glu Ser Ser Leu Pro Ser Ala Ser Lys 630 635 640 acc aaa aag ggc atg ttc cgc acc gtg gga cag ctc tac aaa gag cag 2084 Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gln Leu Tyr Lys Glu Gln 645 650 655 660 ttg ggg aaa ctg atg gct aca ctg cgc aat acc acg gct aac ttt gtg 2132 Leu Gly Lys Leu Met Ala Thr Leu Arg Asn Thr Thr Ala Asn Phe Val 665 670 675 cgc tgc atc atc ccc aac cat gag aag agg tct ggc aag ctg gat gca 2180 Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ser Gly Lys Leu Asp Ala 680 685 690 ttt cta gtg ctg gaa cag ctg cgc tgc aac ggt gtg ttg gaa ggc atc 2228 Phe Leu Val Leu Glu Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile 695 700 705 cgc atc tgc cgt cag ggc ttc ccc aac agg att gtc ttc caa gag ttc 2276 Arg Ile Cys Arg Gln Gly Phe Pro Asn Arg Ile Val Phe Gln Glu Phe 710 715 720 cgg caa cgc tat gag atc ctg gca gcg aac gcc atc ccc aaa ggc ttc 2324 Arg Gln Arg Tyr Glu Ile Leu Ala Ala Asn Ala Ile Pro Lys Gly Phe 725 730 735 740 atg gat gga aag caa gcc tgc att ctc atg atc aaa gcc ctc gaa ctt 2372 Met Asp Gly Lys Gln Ala Cys Ile Leu Met Ile Lys Ala Leu Glu Leu 745 750 755 gac cct aac ctg tac agg att ggg cag agc aaa atc ttc ttc cga acg 2420 Asp Pro Asn Leu Tyr Arg Ile Gly Gln Ser Lys Ile Phe Phe Arg Thr 760 765 770 ggg gtc ctg gcc cac cta gag gag gaa cga gac ctg aaa att act gat 2468 Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu Lys Ile Thr Asp 775 780 785 gtc atc atg gcc ttc cag gca atg tgt cgt ggc tac ctt gcc aga aag 2516 Val Ile Met Ala Phe Gln Ala Met Cys Arg Gly Tyr Leu Ala Arg Lys 790 795 800 gcc ttc acc aag agg cag caa cag ctg aca gcc atg aag gtg atc cag 2564 Ala Phe Thr Lys Arg Gln Gln Gln Leu Thr Ala Met Lys Val Ile Gln 805 810 815 820 agg aac tgc gct gcc tac ctt aag ctc cgc aac tgg caa tgg tgg cgg 2612 Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg Asn Trp Gln Trp Trp Arg 825 830 835 ctc ttc acc aaa gta aag cca ttg ctc cag gtg aca cgg cag gag gag 2660 Leu Phe Thr Lys Val Lys Pro Leu Leu Gln Val Thr Arg Gln Glu Glu 840 845 850 gag atg cag gcc aag gag gag gag atg caa aag atc acg gag cgg cag 2708 Glu Met Gln Ala Lys Glu Glu Glu Met Gln Lys Ile Thr Glu Arg Gln 855 860 865 cag aag gca gag act gag ttg aag gag ctg gag cag aag cac act cag 2756 Gln Lys Ala Glu Thr Glu Leu Lys Glu Leu Glu Gln Lys His Thr Gln 870 875 880 ctg gct gag gag aag act ctg ctg cag gag cag ttg cag gca gag aca 2804 Leu Ala Glu Glu Lys Thr Leu Leu Gln Glu Gln Leu Gln Ala Glu Thr 885 890 895 900 gag ctg tat gct gag tct gag gag atg cgg gtc cgg ttg gca gcc aag 2852 Glu Leu Tyr Ala Glu Ser Glu Glu Met Arg Val Arg Leu Ala Ala Lys 905 910 915 aag cag gaa ctg gag gag atc cta cat gag atg gag gcc cgc ctg gag 2900 Lys Gln Glu Leu Glu Glu Ile Leu His Glu Met Glu Ala Arg Leu Glu 920 925 930 gaa gag gaa gac cgg cgc cag caa cta cag gct gag agg aag aag atg 2948 Glu Glu Glu Asp Arg Arg Gln Gln Leu Gln Ala Glu Arg Lys Lys Met 935 940 945 gct cag cag atg cta gac ctg gag gag caa ctg gag gag gaa gag gcc 2996 Ala Gln Gln Met Leu Asp Leu Glu Glu Gln Leu Glu Glu Glu Glu Ala 950 955 960 gcc aga cag aaa cta cag cta gag aag gtc acg gct gag gcc aag atc 3044 Ala Arg Gln Lys Leu Gln Leu Glu Lys Val Thr Ala Glu Ala Lys Ile 965 970 975 980 aag aaa ctg gag gat gac atc ttg gtt atg gat gat cag aac agt aaa 3092 Lys Lys Leu Glu Asp Asp Ile Leu Val Met Asp Asp Gln Asn Ser Lys 985 990 995 ctt tca aaa gaa cga aaa ctc ctt gaa gag agg gtc agc gac ttg aca 3140 Leu Ser Lys Glu Arg Lys Leu Leu Glu Glu Arg Val Ser Asp Leu Thr 1000 1005 1010 acc aac cta gca gaa gag gaa gaa aag gct aaa aac ctc aca aag ctg 3188 Thr Asn Leu Ala Glu Glu Glu Glu Lys Ala Lys Asn Leu Thr Lys Leu 1015 1020 1025 aag agc aag cat gag tct atg atc tca gag ctg gag gtg agg ctg aag 3236 Lys Ser Lys His Glu Ser Met Ile Ser Glu Leu Glu Val Arg Leu Lys 1030 1035 1040 aaa gag gag aag agc cgg cag gag ctg gag aaa ctc aag agg aaa ctg 3284 Lys Glu Glu Lys Ser Arg Gln Glu Leu Glu Lys Leu Lys Arg Lys Leu 1045 1050 1055 1060 gag ggt gat gcc agt gac ttc cat gag cag atc gct gac ttg cag gcc 3332 Glu Gly Asp Ala Ser Asp Phe His Glu Gln Ile Ala Asp Leu Gln Ala 1065 1070 1075 cag att gca gag ctc aag atg cag ctg gca aag aaa gag gaa gag cta 3380 Gln Ile Ala Glu Leu Lys Met Gln Leu Ala Lys Lys Glu Glu Glu Leu 1080 1085 1090 cag gca gct cta gcc agg ctt gat gaa gag atc gcc cag aaa aac aat 3428 Gln Ala Ala Leu Ala Arg Leu Asp Glu Glu Ile Ala Gln Lys Asn Asn 1095 1100 1105 gcc cta aag aag att cgc gag cta gag ggc cat atc tca gac cta caa 3476 Ala Leu Lys Lys Ile Arg Glu Leu Glu Gly His Ile Ser Asp Leu Gln 1110 1115 1120 gag gac cta gac tca gag cgg gct gcc agg aac aag gcc gag aag cag 3524 Glu Asp Leu Asp Ser Glu Arg Ala Ala Arg Asn Lys Ala Glu Lys Gln 1125 1130 1135 1140 aag cga gac ctg ggg gag gag ctg gag gca ctc aag acg gag ctg gag 3572 Lys Arg Asp Leu Gly Glu Glu Leu Glu Ala Leu Lys Thr Glu Leu Glu 1145 1150 1155 gat acg ctg gac agc aca gct acc cag cag gag ctc aga gcc aag agg 3620 Asp Thr Leu Asp Ser Thr Ala Thr Gln Gln Glu Leu Arg Ala Lys Arg 1160 1165 1170 gaa cag gag gtg aca gtg ctg aag aag gcc ctg gat gag gag acg cgg 3668 Glu Gln Glu Val Thr Val Leu Lys Lys Ala Leu Asp Glu Glu Thr Arg 1175 1180 1185 tcc cat gag gcc cag gtc cag gag atg agg cag aag cac aca cag gca 3716 Ser His Glu Ala Gln Val Gln Glu Met Arg Gln Lys His Thr Gln Ala 1190 1195 1200 gtg gag gaa ctc aca gag cag ctg gag cag ttc aaa agg gcc aag gca 3764 Val Glu Glu Leu Thr Glu Gln Leu Glu Gln Phe Lys Arg Ala Lys Ala 1205 1210 1215 1220 aac ctg gac aaa agc aag cag aca ctg gag aag gag aac gcg gac ctg 3812 Asn Leu Asp Lys Ser Lys Gln Thr Leu Glu Lys Glu Asn Ala Asp Leu 1225 1230 1235 gct ggg gag ctg cgt gtc ctg ggc cag gcg aag cag gag gtg gaa cac 3860 Ala Gly Glu Leu Arg Val Leu Gly Gln Ala Lys Gln Glu Val Glu His 1240 1245 1250 aag aag aag aag ctg gag gtg cag ctg cag gat ctg cag tcc aag tgc 3908 Lys Lys Lys Lys Leu Glu Val Gln Leu Gln Asp Leu Gln Ser Lys Cys 1255 1260 1265 agt gat ggg gag cgt gcc cgg gct gag ctc agt gac aag gtc cac aag 3956 Ser Asp Gly Glu Arg Ala Arg Ala Glu Leu Ser Asp Lys Val His Lys 1270 1275 1280 cta cag aat gaa gtg gag agt gtc act ggc atg ctc aat gag gca gag 4004 Leu Gln Asn Glu Val Glu Ser Val Thr Gly Met Leu Asn Glu Ala Glu 1285 1290 1295 1300 ggc aaa gcc atc aaa ctg gcc aaa gat gtg gct tcc ctt gga tcc cag 4052 Gly Lys Ala Ile Lys Leu Ala Lys Asp Val Ala Ser Leu Gly Ser Gln 1305 1310 1315 ctt cag gac acc caa gag ctg ctc caa gaa gaa acc cgg cag aag ctc 4100 Leu Gln Asp Thr Gln Glu Leu Leu Gln Glu Glu Thr Arg Gln Lys Leu 1320 1325 1330 aat gtg tct acc aag ctg cgt cag ttg gaa gat gaa agg aac agc ctg 4148 Asn Val Ser Thr Lys Leu Arg Gln Leu Glu Asp Glu Arg Asn Ser Leu 1335 1340 1345 cag gac cag ctg gat gag gag atg gag gct aag caa aac ctg gag cgc 4196 Gln Asp Gln Leu Asp Glu Glu Met Glu Ala Lys Gln Asn Leu Glu Arg 1350 1355 1360 cat gtc tca aca ctg aac att cag ctc tca gac tct aag aag aag ctg 4244 His Val Ser Thr Leu Asn Ile Gln Leu Ser Asp Ser Lys Lys Lys Leu 1365 1370 1375 1380 cag gac ttt gca agt acc atc gag gtc atg gag gag ggg aag aag agg 4292 Gln Asp Phe Ala Ser Thr Ile Glu Val Met Glu Glu Gly Lys Lys Arg 1385 1390 1395 tta cag aaa gag atg gag ggc ctc agc cag cag tat gag gag aag gcg 4340 Leu Gln Lys Glu Met Glu Gly Leu Ser Gln Gln Tyr Glu Glu Lys Ala 1400 1405 1410 gct gcc tat gac aaa ctg gag aaa acc aag aac agg ctc cag cag gag 4388 Ala Ala Tyr Asp Lys Leu Glu Lys Thr Lys Asn Arg Leu Gln Gln Glu 1415 1420 1425 ctg gat gac ctg gtc gtg gac ttg gac aac cag cgg caa ctg gta tcc 4436 Leu Asp Asp Leu Val Val Asp Leu Asp Asn Gln Arg Gln Leu Val Ser 1430 1435 1440 aat ctg gaa aag aag cag aag aaa ttt gac cag ttg tta gct gag gag 4484 Asn Leu Glu Lys Lys Gln Lys Lys Phe Asp Gln Leu Leu Ala Glu Glu 1445 1450 1455 1460 aag aac atc tcc tcc aag tat gcg gat gag aga gac cga gct gaa gca 4532 Lys Asn Ile Ser Ser Lys Tyr Ala Asp Glu Arg Asp Arg Ala Glu Ala 1465 1470 1475 gag gcc agg gaa aag gag aca aag gct ttg tct cta gcc cgg gcc ctg 4580 Glu Ala Arg Glu Lys Glu Thr Lys Ala Leu Ser Leu Ala Arg Ala Leu 1480 1485 1490 gag gaa gcc ctg gaa gcc aaa gaa gag ctg gag agg acc aac aag atg 4628 Glu Glu Ala Leu Glu Ala Lys Glu Glu Leu Glu Arg Thr Asn Lys Met 1495 1500 1505 ctc aaa gct gag atg gaa gac ctg gtc agc tcc aag gat gat gta ggc 4676 Leu Lys Ala Glu Met Glu Asp Leu Val Ser Ser Lys Asp Asp Val Gly 1510 1515 1520 aag aac gtg cat gaa ctg gag aag tcc aag cgt gcc ttg gag acc cag 4724 Lys Asn Val His Glu Leu Glu Lys Ser Lys Arg Ala Leu Glu Thr Gln 1525 1530 1535 1540 atg gaa gag atg aaa acc cag ctg gag gag tcg gag gat gac gtg cag 4772 Met Glu Glu Met Lys Thr Gln Leu Glu Glu Ser Glu Asp Asp Val Gln 1545 1550 1555 gcc act gag gat gcc aag ctg cgg cta gag gtc aac atg cag gcc ctc 4820 Ala Thr Glu Asp Ala Lys Leu Arg Leu Glu Val Asn Met Gln Ala Leu 1560 1565 1570 aag ggc cag ttt gaa cgc gat ctc cag gct cgg gat gaa cag aat gag 4868 Lys Gly Gln Phe Glu Arg Asp Leu Gln Ala Arg Asp Glu Gln Asn Glu 1575 1580 1585 gag aag agg agg cag cta cag cgg cag ctg cac gag tat gag aca gaa 4916 Glu Lys Arg Arg Gln Leu Gln Arg Gln Leu His Glu Tyr Glu Thr Glu 1590 1595 1600 ctg gaa gat gaa cgg aag cag aga gct ctg gcg gcg gca gct aag aag 4964 Leu Glu Asp Glu Arg Lys Gln Arg Ala Leu Ala Ala Ala Ala Lys Lys 1605 1610 1615 1620 aag ctg gaa ggg gac cta aaa gac cta gag ctc cag gct gac tca gcc 5012 Lys Leu Glu Gly Asp Leu Lys Asp Leu Glu Leu Gln Ala Asp Ser Ala 1625 1630 1635 atc aaa ggg agg gag gaa gcc atc aag cag ctt cga aaa ctg cag gct 5060 Ile Lys Gly Arg Glu Glu Ala Ile Lys Gln Leu Arg Lys Leu Gln Ala 1640 1645 1650 cag atg aag gac ttc caa aga gag ctg gat gat gcc cgt gcc tcc agg 5108 Gln Met Lys Asp Phe Gln Arg Glu Leu Asp Asp Ala Arg Ala Ser Arg 1655 1660 1665 gat gag atc ttt gcc acc tca aaa gag aat gag aag aaa gcc aag agt 5156 Asp Glu Ile Phe Ala Thr Ser Lys Glu Asn Glu Lys Lys Ala Lys Ser 1670 1675 1680 ctg gag gca gac ctc atg cag ctc caa gag gac ctg gca gca gct gag 5204 Leu Glu Ala Asp Leu Met Gln Leu Gln Glu Asp Leu Ala Ala Ala Glu 1685 1690 1695 1700 aga gct cgc aag caa gct gac ctg gag aag gag gag ctg gcc gag gag 5252 Arg Ala Arg Lys Gln Ala Asp Leu Glu Lys Glu Glu Leu Ala Glu Glu 1705 1710 1715 ctg gct agc agc ttg tca gga agg aat aca ctg cag gat gag aag cgc 5300 Leu Ala Ser Ser Leu Ser Gly Arg Asn Thr Leu Gln Asp Glu Lys Arg 1720 1725 1730 cgc ctg gag gca agg atc gcc caa cta gag gag gag ctg gag gaa gag 5348 Arg Leu Glu Ala Arg Ile Ala Gln Leu Glu Glu Glu Leu Glu Glu Glu 1735 1740 1745 cag ggc aac atg gag gcc atg agt gat aga gta cgc aag gcc aca ctg 5396 Gln Gly Asn Met Glu Ala Met Ser Asp Arg Val Arg Lys Ala Thr Leu 1750 1755 1760 cag gct gag caa ctg agc aat gag ctg gcc aca gaa cgc agc acg gct 5444 Gln Ala Glu Gln Leu Ser Asn Glu Leu Ala Thr Glu Arg Ser Thr Ala 1765 1770 1775 1780 cag aag aat gag agc gca cgg caa cag ctg gag cgc cag aac aag gaa 5492 Gln Lys Asn Glu Ser Ala Arg Gln Gln Leu Glu Arg Gln Asn Lys Glu 1785 1790 1795 ctg cga agc aag ttg cag gag gta gaa ggt gct gtc aaa gcc aag ctc 5540 Leu Arg Ser Lys Leu Gln Glu Val Glu Gly Ala Val Lys Ala Lys Leu 1800 1805 1810 aag tcc act gtt gcg gcg ctg gag gcc aag att gca cag ctg gag gag 5588 Lys Ser Thr Val Ala Ala Leu Glu Ala Lys Ile Ala Gln Leu Glu Glu 1815 1820 1825 cag gtt gaa cag gag gcc aga gag aaa cag gcg gcc acc aag tcg ctg 5636 Gln Val Glu Gln Glu Ala Arg Glu Lys Gln Ala Ala Thr Lys Ser Leu 1830 1835 1840 aag caa aag gac aag aag cta aag gag gtc ctg ctg cag gtg gag gat 5684 Lys Gln Lys Asp Lys Lys Leu Lys Glu Val Leu Leu Gln Val Glu Asp 1845 1850 1855 1860 gag cgc aag atg gca gag cag tac aag gag cag gca gag aaa gga aac 5732 Glu Arg Lys Met Ala Glu Gln Tyr Lys Glu Gln Ala Glu Lys Gly Asn 1865 1870 1875 acc aag gtc aag cag ctg aag agg cag ctg gaa gag gca gag gag gag 5780 Thr Lys Val Lys Gln Leu Lys Arg Gln Leu Glu Glu Ala Glu Glu Glu 1880 1885 1890 tcc cag tgc atc aac gcc aac cgc agg aag ctg cag cgg gag cta gat 5828 Ser Gln Cys Ile Asn Ala Asn Arg Arg Lys Leu Gln Arg Glu Leu Asp 1895 1900 1905 gag gcc aca gag agc aat gag gcc atg ggc cgt gag gtg aac gcc ctc 5876 Glu Ala Thr Glu Ser Asn Glu Ala Met Gly Arg Glu Val Asn Ala Leu 1910 1915 1920 aag agc aaa ctc agg aga gga aac gag gct tca ttt gtt cct tcc aga 5924 Lys Ser Lys Leu Arg Arg Gly Asn Glu Ala Ser Phe Val Pro Ser Arg 1925 1930 1935 1940 agg gct ggg ggc cgt aga gtt att gaa aac aca gat ggt tct gaa gaa 5972 Arg Ala Gly Gly Arg Arg Val Ile Glu Asn Thr Asp Gly Ser Glu Glu 1945 1950 1955 gaa atg gac gct cgg gac tca gac ttc aat gga acc aaa gcc agt gaa 6020 Glu Met Asp Ala Arg Asp Ser Asp Phe Asn Gly Thr Lys Ala Ser Glu 1960 1965 1970 taaattcagg attggacacc atgtcaggga aaacagaaca ctaaacgaca gcagagccca 6080 gcagactgct tagcacttgt gtccattcgt tctcaagtca cagaaatcac tccacccctc 6140 accaggagtc aaccacagcc ctgcacaaag ggtgt 6175 2 5919 DNA Mus musculus misc_feature (1)...(5919) n = A,T,C or G 2 atggcgcaga anggncanct cagcgangat gagaagttcc tctttgtgga naanaacttc 60 atnaacagcc cnntggcnca ggccgactgg gtngccaaga ngctggtgtg ggtcccttcn 120 gagaagcagg gcttcgangc ngccagcatc aaggaggaga agggngatga ggtggtcgtg 180 gagntggtgg anaatggnaa gaaggtcacn gtnggcaang atgacatcca naanatgaac 240 ccncccaagt tctcnaaggt ggangacatg gcngagctga cgtgbctcaa ngangcntcn 300 gtgctgcaca acctgaggga gvgntacttc tcnggnctca tctanacnta ctcnggcctc 360 ttctgbgtgg tggtcaaccc ctacaagyas ctncccatct actcnganaa gatcgtggan 420 atgtacaagg gcaagaagag gcangagatg ccnccncaca tctabgccat ngccgacacn 480 gcctacagna gcatgctnca ngatcgngan gaccagtcca ttctntgcac aggngagtct 540 ggagccggna agacngagaa cacnvagaaa gtcatncagt acntggcngt ggtggcntcn 600 tcccacaagg gcaagaanga cannagcatc acgggggagc tgganaagca gcttctncan 660 gcaaacccna tcctggaggc nttnggcaan gcnaanacng tcaagaanga caactcctcn 720 cgnttnggca agttcatncg catcaacttn gangtcactg gttacatngt nggngccaan 780 attganacnt atctnctgga aaagtcnvgn gcnatncgnc angcnvgnga ngagagnacn 840 ttncacatct tntactacct gntngcnggn gccaaggana agatganaan tgacntgctn 900 ttggagngct tcaacancta cacattnntn tccaatggct ttgtgcccat cccagcngcn 960 cangatgang agatgttcca gganacnntg gangccatgt cnatcatggg cttcantgaa 1020 gaggancagc tnncnntntt gaaggtngtn tcntcngtcc tncagcttgg aaacatngtc 1080 ttcaagaagg anvgaaacac agaccaggcn tccatgccng anaacacagc ngcncagaaa 1140 gtttgccacc tcntgggnat taangtgaca gatttcacna ganccatcct gaccccncgt 1200 atcaaagttg gacgggangt ngtgcagaan gctcagacna aagaacaggc ngacttcgcn 1260 ntcgaggcnt tngcnaaggc cacntatgan cgccttttcc gntggatnct cagccgtgtn 1320 aacaangccn tggacaagac ccatcggcag ggggcntcct tcctgggnat nctgganatn 1380 gctggntttg anatctttga ggtnaactcc ttcgagcagc tgtgcatcaa ctacaccaac 1440 gagaagctgc agcagctgtt caaccacacn atgttcatcc tggagcagga ngagtaccag 1500 cgngagggca tcgagtggaa cttcatcgac ttcggnctng acctgcagcc nwgyattgag 1560 ctnattgagc ggccgaacaa ccctccnggt gtgctggccc tgctggatga ngantgctgg 1620 ttccccaaag cnacaganaa gtcttttgtg gagaagctnt gcncagagca nggnaancac 1680 cccaanttnc agaagcccaa gcagctnaag gacaaaacng agttctccat catccantan 1740 gctgggaagg tggactacaa ngcnagtgcc tggctgacca agaacatgga cccnctnaat 1800 gacaangtga cntcnctcct caangcctcc tcngacaagt tngtggcnga cctntggaag 1860 gangtggacc gcatngtggg gctggaccag atggccaaga tgacngagag ctcactgccc 1920 agngcctcna agaccaanaa gggcatgttc cgcacngtgg gncagctnta caangagcag 1980 ntggggaanc tgatgncnac nctgcgcaan accacgncna acttngtgcg ctgcatcatc 2040 cccaaccang agaagnggtc nggcaagctg gangcnttnc tngtgctgga ncagctgcgn 2100 tgcaacggng tgntggaagg catccgnatc tgccgncagg gcttccccaa caggatngtc 2160 ttccangagt tccgncaacg ctangagatc ctggcagcna acgccatccc caanggcttc 2220 atggatggna agcaagcctg cattctcatg atcaaagcnc tngaactnga cccnaacntg 2280 tacaggatng ggcagagcaa aatcttcttc cgnacgggng tnctggccca cctngaggag 2340 gancgngacn tgaanatnac ngangtcatc atggccttcc aggcnatgtg tcgtggctac 2400 ctngccvgna aggccttcnc caagnggcag cancagctga cngccatgaa ggtgatccag 2460 aggaactgcg cngcctacct naagctncgn aactggcant ggtggcgnct cttcaccaan 2520 gtnaagccnn tgctncaggt gacacggcag gaggaggaga tgcaggccaa ggaggangag 2580 ntgcanaaga tcanggagcg ncagcagaag gcngaganng agntnvagga gctgvagcag 2640 aagcacacnc agctgncnga ggagaagann ctgctgcagg agcagntgca ggcngagacn 2700 gagctgtang cngagncnga ggagatgcgn gtccggntgg cngccaagaa gcagganctg 2760 gagganatcc tncatgagat ggaggcccgc ctggaggang aggaagaccg gngccagcan 2820 ctncaggcng agaggaagaa gatggcncag cagatgctng acctgganga gcaactggag 2880 gaggangang cngccagnca gaanctacag ctnganaagg tcacngcnga ggccaagatc 2940 aagaanntgg aggangacat cntggtnatg gangatcaga acannaanct ntcaaaagan 3000 cgaaaactcc tngaagagag gntnagngan ttnacaacna anctngcnga ngaggaagan 3060 aaggcnaana acctnacnaa gctgaaganc aagcatgant cnatgatctc aganctggan 3120 gtgnggctga agaangagga gaagagccgg caggagctgg agaanctnaa gnggaanntg 3180 ganggngang ccagtgacnt ccangagcag atcgcngacn tncaggcnca gatngcagag 3240 ctcaagatgc agctggcnaa gaangangan gagctncagg cngcnctngc caggctngan 3300 ganganannn cncagaanaa caangccctn aagaagatnc gngagctnga gggncanatc 3360 tcngacctnc angaggacct ngactcagag cgggcngcca ggaacaaggc nganaagcag 3420 aagcgagacc tnggngagga gctggaggcn ctnaagacng agctgganga nacnctggac 3480 ancacngcna cncagcagga gctcvgngcc aagnggganc aggaggtgac ngtgctgaag 3540 aaggccctgg angangagac ncggtcccat gaggcncagg tccaggagat gaggcagaan 3600 cacncacagg nngtggagga nctcacngag cagctnganc agttcaanag ggccaaggcn 3660 aacctngaca anancaagca gacnctggag aangagaacg cngacctggc nggngagctg 3720 cgngtcctgg gccaggcnaa gcaggaggtg gancanaaga agaagaagct ggaggngcag 3780 ntgcagganc tgcagtccaa gtgcagngat ggggagcgng cccgggcnga gctcanngac 3840 aangtccaca agctncagaa tgaagtngag agngtcacng gnatgctnan ngaggcngag 3900 ggnaangcca tnaanctggc caangangtg gcntccctng gntcccagct ncagganacc 3960 cangagctgc tncaagaaga aacccggcag aagctcaang tgtcnacnaa gctgcgncag 4020 ntggangang annggaacag cctgcangan cagctggang aggagatgga ggcnaagcan 4080 aacctggagc gccanntctc nacnctnaac atncagctct cngactcnaa gaagaagctg 4140 caggactttg cnagnaccnt ngannnyntg ganganggna agaagaggtt ncagaangan 4200 atngagnncc tcanccagca gtaygangag aangcngcng cntanganaa actgganaan 4260 accaagaaca ggctncagca ggagctggan gacctggtng tnganttgga naaccagcgg 4320 caactngtnt ccaanctgga aaagaagcag angaanttng ancagttgtt agcngaggan 4380 aanaacatct cntccaanta ngcggatgan agngacvgag cngangcnga ngcnagggan 4440 aagganacna aggcnntgtc nctngcncgg gccctngang angccntgga ngccaaagan 4500 ganctngagv gnaccaacaa natgctcaan gcnganatgg aagacctngt cagctccaag 4560 gangangtng gcaagaacgt ncatganctg gagaagtcca agcgngccnt ggagacncag 4620 atggangaga tgaanacnca gctggangag yyrgaggang anntgcangc cacngaggan 4680 gccaanctgc ggntngangt caacatgcag gcnctcaang nccagttnga nvgngatctc 4740 cangcncggg angancagaa ngaggagaag aggaggcanc tncagvgnca gctncangag 4800 tangagacng aactggaaga ngancgnaag canvgngcnc tggcngcngc agcnaagaag 4860 aagctggang gggacctnaa agacctngag ctncaggcng actcngccat caangggngg 4920 gaggaagcca tcaagcagct ncnnaaactg caggctcaga tgaaggactt ncanagagan 4980 ctggangatg cccgtgcctc cagngangag atctttgcca cnncnaanga gaangagaag 5040 aaagccaaga gnntggangc agacctcatg cagctncaag agganctngc ngcngcngag 5100 agngctcgca ancangcnga cntngagaag gagganctgg cngaggagct ggcnagnagc 5160 ntgtcnggaa ggaanncnct ncaggangag aagcgccgcc tggaggcnng gatcgcncan 5220 ctngaggagg agctggagga ngancagggc aacatggagg cnatgagnga nvgngtncgc 5280 aangcnacnc ngcaggcnga gcanctnagc aangagctgg ccacagancg cagcacngcn 5340 cagaagaatg agagnncncg gcancagctn gagcgncaga acaagganct nmrnagcaag 5400 ntncangagn tnganggngc ngtcaanncc aagntcaant ccacnntngc ggcgctggag 5460 gccaagattg cncagctnga ggagcaggtn gancaggagg ccagagagaa ncaggcggcc 5520 nccaagncgc tgaagcanan ngacaagaag ctnaaggann tnntgctgca ggtggangan 5580 gagcgcaaga tggcngagca gtacaaggag caggcagaga aaggnaannc canggtcaag 5640 cagctnaaga ggcagctgga ngaggcngag gaggagtcnc agngcatcaa cgccaaccgc 5700 aggaagctgc agcgggagct ngangaggcc acngagagca angaggccat gggccgngag 5760 gtgaacgcnc tcaagagcaa nctcaggvga ggaaacgagn cntcnttngt tcctncnaga 5820 aggnctggng gncgtagagt tattgaaaan ncagatggnt ctganganga anbggacnct 5880 cgngacncag acttcaatgg aaccaanncc agtgaatra 5919 3 1972 PRT Mus musculus 3 Met Ala Gln Lys Gly Gln Leu Ser Asp Asp Glu Lys Phe Leu Phe Val 1 5 10 15 Asp Lys Asn Phe Met Asn Ser Pro Met Ala Gln Ala Asp Trp Val Ala 20 25 30 Lys Lys Leu Val Trp Val Pro Ser Glu Lys Gln Gly Phe Glu Ala Ala 35 40 45 Ser Ile Lys Glu Glu Lys Gly Asp Glu Val Val Val Glu Leu Val Glu 50 55 60 Asn Gly Lys Lys Val Thr Val Gly Lys Asp Asp Ile Gln Lys Met Asn 65 70 75 80 Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu 85 90 95 Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Phe Ser Gly 100 105 110 Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Val Asn Pro Tyr 115 120 125 Lys Tyr Leu Pro Ile Tyr Ser Glu Lys Ile Val Asp Met Tyr Lys Gly 130 135 140 Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ala Asp Thr 145 150 155 160 Ala Tyr Arg Ser Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys 165 170 175 Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Gln Lys Val Ile 180 185 190 Gln Tyr Leu Ala Val Val Ala Ser Ser His Lys Gly Lys Lys Asp Ser 195 200 205 Ser Ile Thr Gly Glu Leu Glu Lys Gln Leu Leu Gln Ala Asn Pro Ile 210 215 220 Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser 225 230 235 240 Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile 245 250 255 Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Ile 260 265 270 Arg Gln Ala Arg Asp Glu Arg Thr Phe His Ile Phe Tyr Tyr Leu Leu 275 280 285 Ala Gly Ala Lys Glu Lys Met Lys Ser Asp Leu Leu Leu Glu Ser Phe 290 295 300 Asn Ser Tyr Thr Phe Leu Ser Asn Gly Phe Val Pro Ile Pro Ala Ala 305 310 315 320 Gln Asp Asp Glu Met Phe Gln Glu Thr Leu Glu Ala Met Ser Ile Met 325 330 335 Gly Phe Asn Glu Glu Glu Gln Leu Ala Ile Leu Lys Val Val Ser Ser 340 345 350 Val Leu Gln Leu Gly Asn Ile Val Phe Lys Lys Glu Arg Asn Thr Asp 355 360 365 Gln Ala Ser Met Pro Asp Asn Thr Ala Ala Gln Lys Val Cys His Leu 370 375 380 Val Gly Ile Asn Val Thr Asp Phe Thr Arg Ala Ile Leu Thr Pro Arg 385 390 395 400 Ile Lys Val Gly Arg Asp Val Val Gln Lys Ala Gln Thr Lys Glu Gln 405 410 415 Ala Asp Phe Ala Ile Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu 420 425 430 Phe Arg Trp Ile Leu Ser Arg Val Asn Lys Ala Leu Asp Lys Thr His 435 440 445 Arg Gln Gly Ala Ser Phe Leu Gly Ile Leu Asp Ile Ala Gly Phe Glu 450 455 460 Ile Phe Glu Val Asn Ser Phe Glu Gln Leu Cys Ile Asn Tyr Thr Asn 465 470 475 480 Glu Lys Leu Gln Gln Leu Phe Asn His Thr Met Phe Ile Leu Glu Gln 485 490 495 Glu Glu Tyr Gln Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly 500 505 510 Leu Asp Leu Gln Pro Ser Ile Glu Leu Ile Glu Arg Pro Asn Asn Pro 515 520 525 Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala 530 535 540 Thr Asp Lys Ser Phe Val Glu Lys Leu Cys Ser Glu Gln Gly Asn His 545 550 555 560 Pro Lys Phe Gln Lys Pro Lys Gln Leu Lys Asp Lys Thr Glu Phe Ser 565 570 575 Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Asn Ala Ser Ala Trp Leu 580 585 590 Thr Lys Asn Met Asp Pro Leu Asn Asp Asn Val Thr Ser Leu Leu Asn 595 600 605 Ala Ser Ser Asp Lys Phe Val Ala Asp Leu Trp Lys Asp Val Asp Arg 610 615 620 Ile Val Gly Leu Asp Gln Met Ala Lys Met Thr Glu Ser Ser Leu Pro 625 630 635 640 Ser Ala Ser Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gln Leu 645 650 655 Tyr Lys Glu Gln Leu Gly Lys Leu Met Ala Thr Leu Arg Asn Thr Thr 660 665 670 Ala Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ser Gly 675 680 685 Lys Leu Asp Ala Phe Leu Val Leu Glu Gln Leu Arg Cys Asn Gly Val 690 695 700 Leu Glu Gly Ile Arg Ile Cys Arg Gln Gly Phe Pro Asn Arg Ile Val 705 710 715 720 Phe Gln Glu Phe Arg Gln Arg Tyr Glu Ile Leu Ala Ala Asn Ala Ile 725 730 735 Pro Lys Gly Phe Met Asp Gly Lys Gln Ala Cys Ile Leu Met Ile Lys 740 745 750 Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gln Ser Lys Ile 755 760 765 Phe Phe Arg Thr Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu 770 775 780 Lys Ile Thr Asp Val Ile Met Ala Phe Gln Ala Met Cys Arg Gly Tyr 785 790 795 800 Leu Ala Arg Lys Ala Phe Thr Lys Arg Gln Gln Gln Leu Thr Ala Met 805 810 815 Lys Val Ile Gln Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg Asn Trp 820 825 830 Gln Trp Trp Arg Leu Phe Thr Lys Val Lys Pro Leu Leu Gln Val Thr 835 840 845 Arg Gln Glu Glu Glu Met Gln Ala Lys Glu Glu Glu Met Gln Lys Ile 850 855 860 Thr Glu Arg Gln Gln Lys Ala Glu Thr Glu Leu Lys Glu Leu Glu Gln 865 870 875 880 Lys His Thr Gln Leu Ala Glu Glu Lys Thr Leu Leu Gln Glu Gln Leu 885 890 895 Gln Ala Glu Thr Glu Leu Tyr Ala Glu Ser Glu Glu Met Arg Val Arg 900 905 910 Leu Ala Ala Lys Lys Gln Glu Leu Glu Glu Ile Leu His Glu Met Glu 915 920 925 Ala Arg Leu Glu Glu Glu Glu Asp Arg Arg Gln Gln Leu Gln Ala Glu 930 935 940 Arg Lys Lys Met Ala Gln Gln Met Leu Asp Leu Glu Glu Gln Leu Glu 945 950 955 960 Glu Glu Glu Ala Ala Arg Gln Lys Leu Gln Leu Glu Lys Val Thr Ala 965 970 975 Glu Ala Lys Ile Lys Lys Leu Glu Asp Asp Ile Leu Val Met Asp Asp 980 985 990 Gln Asn Ser Lys Leu Ser Lys Glu Arg Lys Leu Leu Glu Glu Arg Val 995 1000 1005 Ser Asp Leu Thr Thr Asn Leu Ala Glu Glu Glu Glu Lys Ala Lys Asn 1010 1015 1020 Leu Thr Lys Leu Lys Ser Lys His Glu Ser Met Ile Ser Glu Leu Glu 1025 1030 1035 1040 Val Arg Leu Lys Lys Glu Glu Lys Ser Arg Gln Glu Leu Glu Lys Leu 1045 1050 1055 Lys Arg Lys Leu Glu Gly Asp Ala Ser Asp Phe His Glu Gln Ile Ala 1060 1065 1070 Asp Leu Gln Ala Gln Ile Ala Glu Leu Lys Met Gln Leu Ala Lys Lys 1075 1080 1085 Glu Glu Glu Leu Gln Ala Ala Leu Ala Arg Leu Asp Glu Glu Ile Ala 1090 1095 1100 Gln Lys Asn Asn Ala Leu Lys Lys Ile Arg Glu Leu Glu Gly His Ile 1105 1110 1115 1120 Ser Asp Leu Gln Glu Asp Leu Asp Ser Glu Arg Ala Ala Arg Asn Lys 1125 1130 1135 Ala Glu Lys Gln Lys Arg Asp Leu Gly Glu Glu Leu Glu Ala Leu Lys 1140 1145 1150 Thr Glu Leu Glu Asp Thr Leu Asp Ser Thr Ala Thr Gln Gln Glu Leu 1155 1160 1165 Arg Ala Lys Arg Glu Gln Glu Val Thr Val Leu Lys Lys Ala Leu Asp 1170 1175 1180 Glu Glu Thr Arg Ser His Glu Ala Gln Val Gln Glu Met Arg Gln Lys 1185 1190 1195 1200 His Thr Gln Ala Val Glu Glu Leu Thr Glu Gln Leu Glu Gln Phe Lys 1205 1210 1215 Arg Ala Lys Ala Asn Leu Asp Lys Ser Lys Gln Thr Leu Glu Lys Glu 1220 1225 1230 Asn Ala Asp Leu Ala Gly Glu Leu Arg Val Leu Gly Gln Ala Lys Gln 1235 1240 1245 Glu Val Glu His Lys Lys Lys Lys Leu Glu Val Gln Leu Gln Asp Leu 1250 1255 1260 Gln Ser Lys Cys Ser Asp Gly Glu Arg Ala Arg Ala Glu Leu Ser Asp 1265 1270 1275 1280 Lys Val His Lys Leu Gln Asn Glu Val Glu Ser Val Thr Gly Met Leu 1285 1290 1295 Asn Glu Ala Glu Gly Lys Ala Ile Lys Leu Ala Lys Asp Val Ala Ser 1300 1305 1310 Leu Gly Ser Gln Leu Gln Asp Thr Gln Glu Leu Leu Gln Glu Glu Thr 1315 1320 1325 Arg Gln Lys Leu Asn Val Ser Thr Lys Leu Arg Gln Leu Glu Asp Glu 1330 1335 1340 Arg Asn Ser Leu Gln Asp Gln Leu Asp Glu Glu Met Glu Ala Lys Gln 1345 1350 1355 1360 Asn Leu Glu Arg His Val Ser Thr Leu Asn Ile Gln Leu Ser Asp Ser 1365 1370 1375 Lys Lys Lys Leu Gln Asp Phe Ala Ser Thr Ile Glu Val Met Glu Glu 1380 1385 1390 Gly Lys Lys Arg Leu Gln Lys Glu Met Glu Gly Leu Ser Gln Gln Tyr 1395 1400 1405 Glu Glu Lys Ala Ala Ala Tyr Asp Lys Leu Glu Lys Thr Lys Asn Arg 1410 1415 1420 Leu Gln Gln Glu Leu Asp Asp Leu Val Val Asp Leu Asp Asn Gln Arg 1425 1430 1435 1440 Gln Leu Val Ser Asn Leu Glu Lys Lys Gln Lys Lys Phe Asp Gln Leu 1445 1450 1455 Leu Ala Glu Glu Lys Asn Ile Ser Ser Lys Tyr Ala Asp Glu Arg Asp 1460 1465 1470 Arg Ala Glu Ala Glu Ala Arg Glu Lys Glu Thr Lys Ala Leu Ser Leu 1475 1480 1485 Ala Arg Ala Leu Glu Glu Ala Leu Glu Ala Lys Glu Glu Leu Glu Arg 1490 1495 1500 Thr Asn Lys Met Leu Lys Ala Glu Met Glu Asp Leu Val Ser Ser Lys 1505 1510 1515 1520 Asp Asp Val Gly Lys Asn Val His Glu Leu Glu Lys Ser Lys Arg Ala 1525 1530 1535 Leu Glu Thr Gln Met Glu Glu Met Lys Thr Gln Leu Glu Glu Ser Glu 1540 1545 1550 Asp Asp Val Gln Ala Thr Glu Asp Ala Lys Leu Arg Leu Glu Val Asn 1555 1560 1565 Met Gln Ala Leu Lys Gly Gln Phe Glu Arg Asp Leu Gln Ala Arg Asp 1570 1575 1580 Glu Gln Asn Glu Glu Lys Arg Arg Gln Leu Gln Arg Gln Leu His Glu 1585 1590 1595 1600 Tyr Glu Thr Glu Leu Glu Asp Glu Arg Lys Gln Arg Ala Leu Ala Ala 1605 1610 1615 Ala Ala Lys Lys Lys Leu Glu Gly Asp Leu Lys Asp Leu Glu Leu Gln 1620 1625 1630 Ala Asp Ser Ala Ile Lys Gly Arg Glu Glu Ala Ile Lys Gln Leu Arg 1635 1640 1645 Lys Leu Gln Ala Gln Met Lys Asp Phe Gln Arg Glu Leu Asp Asp Ala 1650 1655 1660 Arg Ala Ser Arg Asp Glu Ile Phe Ala Thr Ser Lys Glu Asn Glu Lys 1665 1670 1675 1680 Lys Ala Lys Ser Leu Glu Ala Asp Leu Met Gln Leu Gln Glu Asp Leu 1685 1690 1695 Ala Ala Ala Glu Arg Ala Arg Lys Gln Ala Asp Leu Glu Lys Glu Glu 1700 1705 1710 Leu Ala Glu Glu Leu Ala Ser Ser Leu Ser Gly Arg Asn Thr Leu Gln 1715 1720 1725 Asp Glu Lys Arg Arg Leu Glu Ala Arg Ile Ala Gln Leu Glu Glu Glu 1730 1735 1740 Leu Glu Glu Glu Gln Gly Asn Met Glu Ala Met Ser Asp Arg Val Arg 1745 1750 1755 1760 Lys Ala Thr Leu Gln Ala Glu Gln Leu Ser Asn Glu Leu Ala Thr Glu 1765 1770 1775 Arg Ser Thr Ala Gln Lys Asn Glu Ser Ala Arg Gln Gln Leu Glu Arg 1780 1785 1790 Gln Asn Lys Glu Leu Arg Ser Lys Leu Gln Glu Val Glu Gly Ala Val 1795 1800 1805 Lys Ala Lys Leu Lys Ser Thr Val Ala Ala Leu Glu Ala Lys Ile Ala 1810 1815 1820 Gln Leu Glu Glu Gln Val Glu Gln Glu Ala Arg Glu Lys Gln Ala Ala 1825 1830 1835 1840 Thr Lys Ser Leu Lys Gln Lys Asp Lys Lys Leu Lys Glu Val Leu Leu 1845 1850 1855 Gln Val Glu Asp Glu Arg Lys Met Ala Glu Gln Tyr Lys Glu Gln Ala 1860 1865 1870 Glu Lys Gly Asn Thr Lys Val Lys Gln Leu Lys Arg Gln Leu Glu Glu 1875 1880 1885 Ala Glu Glu Glu Ser Gln Cys Ile Asn Ala Asn Arg Arg Lys Leu Gln 1890 1895 1900 Arg Glu Leu Asp Glu Ala Thr Glu Ser Asn Glu Ala Met Gly Arg Glu 1905 1910 1915 1920 Val Asn Ala Leu Lys Ser Lys Leu Arg Arg Gly Asn Glu Ala Ser Phe 1925 1930 1935 Val Pro Ser Arg Arg Ala Gly Gly Arg Arg Val Ile Glu Asn Thr Asp 1940 1945 1950 Gly Ser Glu Glu Glu Met Asp Ala Arg Asp Ser Asp Phe Asn Gly Thr 1955 1960 1965 Lys Ala Ser Glu 1970 4 1972 PRT Oryctolagus cuniculus 4 Met Ala Gln Lys Gly Gln Leu Ser Asp Asp Glu Lys Phe Leu Phe Val 1 5 10 15 Asp Lys Asn Phe Ile Asn Ser Pro Val Ala Gln Ala Asp Trp Val Ala 20 25 30 Lys Arg Leu Val Trp Val Pro Ser Glu Lys Gln Gly Phe Glu Ala Ala 35 40 45 Ser Ile Lys Glu Glu Lys Gly Asp Glu Val Val Val Glu Leu Val Glu 50 55 60 Asn Gly Lys Lys Val Thr Val Gly Lys Asp Asp Ile Gln Lys Met Asn 65 70 75 80 Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu 85 90 95 Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Phe Ser Gly 100 105 110 Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Val Asn Pro Tyr 115 120 125 Lys Gln Leu Pro Ile Tyr Ser Glu Lys Ile Val Asp Met Tyr Lys Gly 130 135 140 Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ala Asp Thr 145 150 155 160 Ala Tyr Arg Ser Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys 165 170 175 Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile 180 185 190 Gln Tyr Leu Ala Val Val Ala Ser Ser His Lys Gly Lys Lys Asp Thr 195 200 205 Ser Ile Thr Gly Glu Leu Glu Lys Gln Leu Leu Gln Ala Asn Pro Ile 210 215 220 Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser 225 230 235 240 Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile 245 250 255 Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Ile 260 265 270 Arg Gln Ala Arg Glu Glu Arg Thr Phe His Ile Phe Tyr Tyr Leu Ile 275 280 285 Ala Gly Ala Lys Glu Lys Met Arg Asn Asp Leu Leu Leu Glu Gly Phe 290 295 300 Asn Asn Tyr Thr Phe Leu Ser Asn Gly Phe Val Pro Ile Pro Ala Ala 305 310 315 320 Gln Asp Asp Glu Met Phe Gln Glu Thr Val Glu Ala Met Ser Ile Met 325 330 335 Gly Phe Ser Glu Glu Glu Gln Leu Ser Val Leu Lys Val Val Ser Ser 340 345 350 Val Leu Gln Leu Gly Asn Ile Val Phe Lys Lys Glu Arg Asn Thr Asp 355 360 365 Gln Ala Ser Met Pro Asp Asn Thr Ala Ala Gln Lys Val Cys His Leu 370 375 380 Met Gly Ile Asn Val Thr Asp Phe Thr Arg Ser Ile Leu Thr Pro Arg 385 390 395 400 Ile Lys Val Gly Arg Asp Val Val Gln Lys Ala Gln Thr Lys Glu Gln 405 410 415 Ala Asp Phe Ala Val Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu 420 425 430 Phe Arg Trp Ile Leu Ser Arg Val Asn Lys Ala Leu Asp Lys Thr His 435 440 445 Arg Gln Gly Ala Ser Phe Leu Gly Ile Leu Asp Ile Ala Gly Phe Glu 450 455 460 Ile Phe Glu Val Asn Ser Phe Glu Gln Leu Cys Ile Asn Tyr Thr Asn 465 470 475 480 Glu Lys Leu Gln Gln Leu Phe Asn His Thr Met Phe Ile Leu Glu Gln 485 490 495 Glu Glu Tyr Gln Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly 500 505 510 Leu Asp Leu Gln Pro Cys Ile Glu Leu Ile Glu Arg Pro Asn Asn Pro 515 520 525 Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala 530 535 540 Thr Asp Lys Ser Phe Val Glu Lys Leu Cys Thr Glu Gln Gly Asn His 545 550 555 560 Pro Lys Phe Gln Lys Pro Lys Gln Leu Lys Asp Lys Thr Glu Phe Ser 565 570 575 Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Asn Ala Ser Ala Trp Leu 580 585 590 Thr Lys Asn Met Asp Pro Leu Asn Asp Asn Val Thr Ser Leu Leu Asn 595 600 605 Ala Ser Ser Asp Lys Phe Val Ala Asp Leu Trp Lys Asp Val Asp Arg 610 615 620 Ile Val Gly Leu Asp Gln Met Ala Lys Met Thr Glu Ser Ser Leu Pro 625 630 635 640 Ser Ala Ser Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gln Leu 645 650 655 Tyr Lys Glu Gln Leu Gly Lys Leu Met Thr Thr Leu Arg Asn Thr Thr 660 665 670 Pro Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ser Gly 675 680 685 Lys Leu Asp Ala Phe Leu Val Leu Glu Gln Leu Arg Cys Asn Gly Val 690 695 700 Leu Glu Gly Ile Arg Ile Cys Arg Gln Gly Phe Pro Asn Arg Ile Val 705 710 715 720 Phe Gln Glu Phe Arg Gln Arg Tyr Glu Ile Leu Ala Ala Asn Ala Ile 725 730 735 Pro Lys Gly Phe Met Asp Gly Lys Gln Ala Cys Ile Leu Met Ile Lys 740 745 750 Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gln Ser Lys Ile 755 760 765 Phe Phe Arg Thr Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu 770 775 780 Lys Ile Thr Asp Val Ile Met Ala Phe Gln Ala Met Cys Arg Gly Tyr 785 790 795 800 Leu Ala Arg Lys Ala Phe Ala Lys Arg Gln Gln Gln Leu Thr Ala Met 805 810 815 Lys Val Ile Gln Arg Asn Cys Ala Ala Tyr Leu Lys Leu Arg Asn Trp 820 825 830 Gln Trp Trp Arg Leu Phe Thr Lys Val Lys Pro Leu Leu Gln Val Thr 835 840 845 Arg Gln Glu Glu Glu Met Gln Ala Lys Glu Asp Glu Leu Gln Lys Ile 850 855 860 Lys Glu Arg Gln Gln Lys Ala Glu Ser Glu Leu Gln Glu Leu Gln Gln 865 870 875 880 Lys His Thr Gln Leu Ser Glu Glu Lys Asn Leu Leu Gln Glu Gln Leu 885 890 895 Gln Ala Glu Thr Glu Leu Tyr Ala Glu Ala Glu Glu Met Arg Val Arg 900 905 910 Leu Ala Ala Lys Lys Gln Glu Leu Glu Glu Ile Leu His Glu Met Glu 915 920 925 Ala Arg Leu Glu Glu Glu Glu Asp Arg Gly Gln Gln Leu Gln Ala Glu 930 935 940 Arg Lys Lys Met Ala Gln Gln Met Leu Asp Leu Glu Glu Gln Leu Glu 945 950 955 960 Glu Glu Glu Ala Ala Arg Gln Lys Leu Gln Leu Glu Lys Val Thr Ala 965 970 975 Glu Ala Lys Ile Lys Lys Leu Glu Asp Asp Ile Leu Val Met Asp Asp 980 985 990 Gln Asn Asn Lys Leu Ser Lys Glu Arg Lys Leu Leu Glu Glu Arg Ile 995 1000 1005 Ser Asp Leu Thr Thr Asn Leu Ala Glu Glu Glu Glu Lys Ala Lys Asn 1010 1015 1020 Leu Thr Lys Leu Lys Asn Lys His Glu Ser Met Ile Ser Glu Leu Glu 1025 1030 1035 1040 Val Arg Leu Lys Lys Glu Glu Lys Ser Arg Gln Glu Leu Glu Lys Leu 1045 1050 1055 Lys Arg Lys Met Asp Gly Glu Ala Ser Asp Leu His Glu Gln Ile Ala 1060 1065 1070 Asp Leu Gln Ala Gln Ile Ala Glu Leu Lys Met Gln Leu Ala Lys Lys 1075 1080 1085 Glu Glu Glu Leu Gln Ala Ala Leu Ala Arg Leu Glu Asp Glu Thr Ser 1090 1095 1100 Gln Lys Asn Asn Ala Leu Lys Lys Ile Arg Glu Leu Glu Gly His Ile 1105 1110 1115 1120 Ser Asp Leu Gln Glu Asp Leu Asp Ser Glu Arg Ala Ala Arg Asn Lys 1125 1130 1135 Ala Glu Lys Gln Lys Arg Asp Leu Gly Glu Glu Leu Glu Ala Leu Lys 1140 1145 1150 Thr Glu Leu Glu Asp Thr Leu Asp Thr Thr Ala Thr Gln Gln Glu Leu 1155 1160 1165 Arg Ala Lys Arg Glu Gln Glu Val Thr Val Leu Lys Lys Ala Leu Asp 1170 1175 1180 Glu Glu Thr Arg Ser His Glu Ala Gln Val Gln Glu Met Arg Gln Lys 1185 1190 1195 1200 His Thr Gln Val Val Glu Glu Leu Thr Glu Gln Leu Glu Gln Phe Lys 1205 1210 1215 Arg Ala Lys Ala Asn Leu Asp Lys Thr Lys Gln Thr Leu Glu Lys Glu 1220 1225 1230 Asn Ala Asp Leu Ala Gly Glu Leu Arg Val Leu Gly Gln Ala Lys Gln 1235 1240 1245 Glu Val Glu His Lys Lys Lys Lys Leu Glu Val Gln Leu Gln Glu Leu 1250 1255 1260 Gln Ser Lys Cys Ser Asp Gly Glu Arg Ala Arg Ala Glu Leu Asn Asp 1265 1270 1275 1280 Lys Val His Lys Leu Gln Asn Glu Val Glu Ser Val Thr Gly Met Leu 1285 1290 1295 Ser Glu Ala Glu Gly Lys Ala Ile Lys Leu Ala Lys Glu Val Ala Ser 1300 1305 1310 Leu Gly Ser Gln Leu Gln Asp Thr Gln Glu Leu Leu Gln Glu Glu Thr 1315 1320 1325 Arg Gln Lys Leu Asn Val Ser Thr Lys Leu Arg Gln Leu Glu Asp Glu 1330 1335 1340 Arg Asn Ser Leu Gln Glu Gln Leu Asp Glu Glu Met Glu Ala Lys Gln 1345 1350 1355 1360 Asn Leu Glu Arg His Ile Ser Thr Leu Asn Ile Gln Leu Ser Asp Ser 1365 1370 1375 Lys Lys Lys Leu Gln Asp Phe Ala Ser Thr Val Glu Ser Leu Glu Glu 1380 1385 1390 Gly Lys Lys Arg Phe Gln Lys Glu Ile Glu Ser Leu Thr Gln Gln Tyr 1395 1400 1405 Glu Glu Lys Ala Ala Ala Tyr Asp Lys Leu Glu Lys Thr Lys Asn Arg 1410 1415 1420 Leu Gln Gln Glu Leu Asp Asp Leu Val Val Asp Leu Asp Asn Gln Arg 1425 1430 1435 1440 Gln Leu Val Ser Asn Leu Glu Lys Lys Gln Lys Lys Phe Asp Gln Leu 1445 1450 1455 Leu Ala Glu Glu Lys Asn Ile Ser Ser Lys Tyr Ala Asp Glu Arg Asp 1460 1465 1470 Arg Ala Glu Ala Glu Ala Arg Glu Lys Glu Thr Lys Ala Leu Ser Leu 1475 1480 1485 Ala Arg Ala Leu Glu Glu Ala Leu Glu Ala Lys Glu Glu Leu Glu Arg 1490 1495 1500 Thr Asn Lys Met Leu Lys Ala Glu Met Glu Asp Leu Val Ser Ser Lys 1505 1510 1515 1520 Asp Asp Val Gly Lys Asn Val His Glu Leu Glu Lys Ser Lys Arg Ala 1525 1530 1535 Leu Glu Thr Gln Met Glu Glu Met Lys Thr Gln Leu Glu Glu Leu Glu 1540 1545 1550 Asp Glu Leu Gln Ala Thr Glu Asp Ala Lys Leu Arg Leu Glu Val Asn 1555 1560 1565 Met Gln Ala Leu Lys Val Gln Phe Glu Arg Asp Leu Gln Ala Arg Asp 1570 1575 1580 Glu Gln Asn Glu Glu Lys Arg Arg Gln Leu Gln Arg Gln Leu His Glu 1585 1590 1595 1600 Tyr Glu Thr Glu Leu Glu Asp Glu Arg Lys Gln Arg Ala Leu Ala Ala 1605 1610 1615 Ala Ala Lys Lys Lys Leu Glu Gly Asp Leu Lys Asp Leu Glu Leu Gln 1620 1625 1630 Ala Asp Ser Ala Ile Lys Gly Arg Glu Glu Ala Ile Lys Gln Leu Leu 1635 1640 1645 Lys Leu Gln Ala Gln Met Lys Asp Phe Gln Arg Glu Leu Glu Asp Ala 1650 1655 1660 Arg Ala Ser Arg Asp Glu Ile Phe Ala Thr Ala Lys Glu Asn Glu Lys 1665 1670 1675 1680 Lys Ala Lys Ser Leu Glu Ala Asp Leu Met Gln Leu Gln Glu Asp Leu 1685 1690 1695 Ala Ala Ala Glu Arg Ala Arg Lys Gln Ala Asp Leu Glu Lys Glu Glu 1700 1705 1710 Leu Ala Glu Glu Leu Ala Ser Ser Leu Ser Gly Arg Asn Ala Leu Gln 1715 1720 1725 Asp Glu Lys Arg Arg Leu Glu Ala Arg Ile Ala Gln Leu Glu Glu Glu 1730 1735 1740 Leu Glu Glu Glu Gln Gly Asn Met Glu Ala Met Ser Asp Arg Val Arg 1745 1750 1755 1760 Lys Ala Thr Gln Gln Ala Glu Gln Leu Ser Asn Glu Leu Ala Thr Glu 1765 1770 1775 Arg Ser Thr Ala Gln Lys Asn Glu Ser Ala Arg Gln Gln Leu Glu Arg 1780 1785 1790 Gln Asn Lys Glu Leu Lys Ser Lys Leu Gln Glu Met Glu Gly Ala Val 1795 1800 1805 Lys Ser Lys Phe Lys Ser Thr Ile Ala Ala Leu Glu Ala Lys Ile Ala 1810 1815 1820 Gln Leu Glu Glu Gln Val Glu Gln Glu Ala Arg Glu Lys Gln Ala Ala 1825 1830 1835 1840 Ala Lys Ala Leu Lys Gln Arg Asp Lys Lys Leu Lys Glu Met Leu Leu 1845 1850 1855 Gln Val Glu Asp Glu Arg Lys Met Ala Glu Gln Tyr Lys Glu Gln Ala 1860 1865 1870 Glu Lys Gly Asn Ala Lys Val Lys Gln Leu Lys Arg Gln Leu Glu Glu 1875 1880 1885 Ala Glu Glu Glu Ser Gln Arg Ile Asn Ala Asn Arg Arg Lys Leu Gln 1890 1895 1900 Arg Glu Leu Asp Glu Ala Thr Glu Ser Asn Glu Ala Met Gly Arg Glu 1905 1910 1915 1920 Val Asn Ala Leu Lys Ser Lys Leu Arg Arg Gly Asn Glu Thr Ser Phe 1925 1930 1935 Val Pro Thr Arg Arg Ser Gly Gly Arg Arg Val Ile Glu Asn Ala Asp 1940 1945 1950 Gly Ser Glu Glu Glu Val Asp Ala Arg Asp Ala Asp Phe Asn Gly Thr 1955 1960 1965 Lys Ser Ser Glu 1970 5 6644 DNA Oryctolagus cuniculus 5 agccttggac gccccggcct gggaggtgtg ccagacccgc gctccccgtc cagtttctcc 60 gcgcgccccc cacttggagg ggaccaacca ggcaccatgg cgcagaaggg ccaactcagc 120 gacgatgaga agttcctctt tgtggacaag aacttcatca acagccccgt ggcccaggcc 180 gactgggtgg ccaagaggct ggtgtgggtc ccttcggaga agcagggctt cgaggcggcc 240 agcatcaagg aggagaaggg ggatgaggtg gtcgtggagc tggtggagaa tgggaagaag 300 gtcacggtgg gcaaggatga catccagaag atgaacccgc ccaagttctc caaggtggaa 360 gacatggcgg agctgacgtg tctcaacgaa gcttccgtgc tgcacaacct gagggagagg 420 tacttctctg ggctcatcta cacgtactcc ggcctcttct gcgtggtggt caacccctac 480 aagcagctgc ccatctactc ggagaagatc gtggacatgt acaagggcaa gaagaggcac 540 gagatgccac cacacatcta cgccatcgcc gacacggcct acaggagcat gctgcaggat 600 cgggaggacc agtccattct ctgcacaggc gagtctggag ccgggaagac ggagaacacc 660 aagaaagtca tccagtacct ggccgtggtg gcctcctcgc acaagggcaa gaaggacacg 720 agcatcacgg gggagctgga gaagcagctt ctgcaagcaa accccatcct ggaggccttt 780 ggcaacgcca agacggtcaa gaatgacaac tcctcgcgat tcggcaagtt catccgcatc 840 aactttgacg tcactggtta catcgtgggc gccaacattg agacctatct gctggaaaag 900 tcacgagcca tccgccaagc ccgagaggag aggaccttcc acatctttta ctacctgatt 960 gctggggcca aggagaagat gagaaatgac ttgctcttgg agggcttcaa caactacaca 1020 ttcctctcca atggctttgt gcccatccca gccgcccagg atgacgagat gttccaggaa 1080 acggtggagg ccatgtccat catgggcttc agtgaagagg agcagctgtc tgtgttgaag 1140 gtggtgtctt cagtcctgca gcttggaaac atcgtcttca agaaggaaag aaacacagac 1200 caggcgtcca tgccggacaa cacagctgcc cagaaagttt gccacctcat gggaattaac 1260 gtgacagatt tcaccagatc catcctgacc ccgcgtatca aagttggacg ggacgtagtg 1320 cagaaagctc agacaaaaga acaggcagac ttcgctgtcg aggctttggc aaaggccacg 1380 tatgaacgcc ttttccgctg gatcctcagc cgtgtgaaca aagccctgga caagacccat 1440 cggcaggggg cttccttcct ggggatcctg gacatcgctg gatttgagat ctttgaggtg 1500 aactccttcg agcagctgtg catcaactac accaacgaga agctgcagca gctgttcaac 1560 cacaccatgt tcatcctgga gcaggaggag taccagcgcg agggcatcga gtggaacttc 1620 atcgacttcg ggctcgacct gcagccctgc attgagctca ttgagcggcc gaacaaccct 1680 ccaggtgtgc tggccctgct ggatgaggag tgctggttcc ccaaagccac agataagtct 1740 tttgtggaga agctgtgcac agagcaaggc aaccacccca agttccagaa gcccaagcag 1800 ctcaaggaca aaacggagtt ctccatcatc cattacgctg ggaaggtgga ctacaacgcg 1860 agtgcctggc tgaccaagaa catggacccc ctgaatgaca acgtgacctc cctcctcaac 1920 gcctcctcgg acaagttcgt ggccgaccta tggaaggacg tggaccgcat cgtggggctg 1980 gaccagatgg ccaagatgac agagagctca ctgcccagcg cctccaagac caagaagggc 2040 atgttccgca cggtggggca gctgtacaag gagcagctgg ggaagctgat gaccacgctg 2100 cgcaacacca cgcccaactt cgtgcgctgc atcatcccca accacgagaa gcggtccggc 2160 aagctggacg cgttcctggt gctggagcag ctgcggtgca acggggtgct ggaaggcatc 2220 cgaatctgcc gccagggctt ccccaacagg atcgtcttcc aggagttccg ccaacgctac 2280 gagatcctgg cagccaacgc catccccaag ggcttcatgg atgggaagca agcctgcatt 2340 ctcatgatca aagctctgga actggacccc aacttgtaca ggatcgggca gagcaaaatc 2400 ttcttccgca cgggcgtgct ggcccacctg gaggaggagc gggacttgaa gatcaccgac 2460 gtcatcatgg ccttccaggc catgtgtcgt ggctacctcg cccgcaaggc cttcgccaag 2520 cggcagcagc agctgaccgc catgaaggtg atccagagga actgcgccgc ctacctgaag 2580 ctgcggaact ggcagtggtg gcgcctcttc accaaggtga agccgctgct gcaggtgaca 2640 cggcaggagg aggagatgca ggccaaggag gatgagctgc agaagatcaa ggagcgacag 2700 cagaaggcgg agagcgagct ccaggagctg cagcagaagc acacgcagct gtccgaggag 2760 aagaacctgc tgcaggagca gctgcaggcg gagacggagc tgtacgcgga ggccgaggag 2820 atgcgcgtcc ggctggcggc caagaagcag gagctggagg aaatcctgca tgagatggag 2880 gcccgcctgg aggaggagga agaccggggc cagcagctgc aggccgagag gaagaagatg 2940 gcccagcaga tgctggacct ggaagagcaa ctggaggagg aggaagctgc caggcagaag 3000 ctacagctcg aaaaggtcac cgccgaggcc aagatcaaga agttggagga cgacatcctg 3060 gtcatggacg atcagaacaa caagctctca aaagagcgaa aactcctgga agagaggatt 3120 agtgatttaa caacaaatct tgccgaggag gaagagaagg ccaagaacct gaccaagctg 3180 aagaacaagc atgaatccat gatctcagaa ctggaagtgc ggctgaagaa ggaggagaag 3240 agccggcagg agctggagaa gctgaagcgg aagatggacg gcgaggccag tgacctccac 3300 gagcagatcg ccgacctcca ggcgcagatc gcagagctca agatgcagct ggccaagaag 3360 gaagaggagc tgcaggcggc cctggccagg ctggaggatg aaacgtctca gaagaacaac 3420 gccctgaaga agatccggga gctggagggg cacatctccg acctgcagga ggacctggac 3480 tcagagcggg ccgccaggaa caaggccgag aagcagaagc gagacctggg ggaggagctg 3540 gaggcgctga agacggagct ggaggacacg ctggacacca cggccaccca gcaggagctc 3600 cgggccaagc gggagcagga ggtgacggtg ctgaagaagg ccctggacga ggagacccgg 3660 tcccatgagg cccaggtcca ggagatgagg cagaaacaca cacaggtggt ggaggagctc 3720 acggagcagc tggaacagtt caagagggcc aaggcgaacc tcgacaagac caagcagacg 3780 ctggagaagg agaacgcaga cctggccggc gagctgcggg tcctgggcca ggccaagcag 3840 gaggtggagc acaagaagaa gaagctggag gtgcagctgc aggagctgca gtccaagtgc 3900 agcgatgggg agcgggcccg ggcggagctc aacgacaagg tccacaagct gcagaatgaa 3960 gtggagagcg tcacgggcat gctcagcgag gccgagggga aggccatcaa gctggccaag 4020 gaggtggcgt ccctcgggtc ccagctccag gatacccagg agctgctcca agaagaaacc 4080 cggcagaagc tcaacgtgtc caccaagctg cggcagctgg aggacgagag gaacagcctg 4140 caggagcagc tggacgagga gatggaggcc aagcagaacc tggagcgcca catctccacc 4200 ctgaacatcc agctctccga ctcaaagaag aagctgcagg actttgccag caccgtggag 4260 tccttggagg aaggcaagaa gaggttccag aaggaaattg agagcctcac ccagcagtac 4320 gaagagaaag cagctgctta cgataaactg gaaaagacca agaacaggct tcagcaggag 4380 ctggacgacc tggtcgtaga cttggataac cagcggcaac tggtgtccaa cctggaaaag 4440 aagcagaaga agttcgatca gttgttagcc gaggaaaaga acatctcttc caagtatgcg 4500 gatgaaaggg accgagccga ggctgaagca agggaaaagg aaaccaaggc cttgtccctg 4560 gctcgggccc tcgaggaggc cttggaggcc aaagaggagc tcgagagaac caacaaaatg 4620 ctcaaggccg agatggaaga cctcgtcagc tccaaggacg acgtgggcaa gaacgtccat 4680 gagctggaga agtccaagcg ggccctggag acacagatgg aggagatgaa gacgcagctg 4740 gaagagctag aggacgagct gcaggccacc gaggacgcca agctgcggtt ggaggtcaac 4800 atgcaggccc tcaaagtcca gttcgagcgg gatctccagg cccgggatga gcagaacgag 4860 gagaagagga ggcagctgca gaggcagctg catgagtacg agacggaact ggaagacgag 4920 cgcaagcagc gggccctggc cgcggcagcc aagaagaagc tggaggggga cctgaaagac 4980 ctggagcttc aggcggactc cgccatcaaa gggcgggagg aagccatcaa gcagcttctg 5040 aaactgcagg ctcagatgaa ggacttccag agagaactgg aagatgcccg tgcctccaga 5100 gacgagatct ttgccacagc caaggagaac gagaagaaag ccaagagtct ggaggcagac 5160 ctcatgcagc tacaagagga tctggccgcg gcagagaggg ctcgcaaaca ggcagacttg 5220 gagaaggagg agctggccga ggagctggcc agcagcttgt ccggaaggaa cgcgctgcag 5280 gatgagaagc gccgcctgga ggcccggatc gcacagctgg aggaggagct ggaggaggaa 5340 cagggcaaca tggaggcaat gagcgaccgc gtccgcaagg ctacgcagca ggccgagcag 5400 ctcagcaacg agctggccac agagcgcagc acagcccaga agaatgagag cgcacggcag 5460 cagctcgagc ggcagaacaa ggagctcaag agcaagctgc aggagatgga gggggcagtc 5520 aagtccaagt tcaagtccac tatcgcggcg ctggaggcca agattgcgca gctggaggag 5580 caggttgagc aggaggccag agagaagcag gcggccgcca aggcgctgaa gcagagggac 5640 aagaagctga aggagatgct gctgcaggtg gaagacgagc gcaagatggc tgagcagtac 5700 aaggagcagg cagagaaagg aaacgccaag gtcaagcagc tcaagaggca gctggaggag 5760 gccgaggagg agtcgcagcg catcaacgcc aaccgcagga agctgcagcg ggagctggac 5820 gaggccacgg agagcaacga ggccatgggc cgcgaggtga acgcgctcaa gagcaagctc 5880 aggcgaggaa acgagacctc gttcgttcct accagaaggt ctggagggcg tagagttatt 5940 gaaaacgcag atggctctga ggaggaagtg gacgctcgcg acgcagactt caatggaacc 6000 aaatccagtg aatgaacgac ttcgagtttt gcaccacagc agaaggtccc accaaagaac 6060 agattcaacc aaacccaacc cagcaaaccc tacttagcat cgtccaaccc tgtcttcaag 6120 tttcaaacat cacggacaac cccagaacat aaaaaccact gcctgagtca gggtacagaa 6180 ttccaagttt ttatgatgtg gcagaggaaa aagccaacag gaacaaaaac accctcgctc 6240 taccgatcag gcagagatgt taagtttttt ggaagataca gccacatgaa ccggtcactg 6300 gtcaattcgt ttacatgaca tgggtgaagt tcaccacgat gggtgccgct gccccactcc 6360 cactcccagt tcattcccta acctctgtgc ccttccaagc ccctgagtaa ggccctggtt 6420 tgagaatccc ttctccctct cctggctacc acacctcaga catgcacagt tcaccccgcc 6480 caagtgcctt ctgtagtcac aagaagtaaa aaaggagaca ctgttccatg gatgagaaca 6540 gggacggtcc actgtcttat gtgcacccaa ttgtacttcg gacaccttca ctaataaaag 6600 gtcatacgtc aaaaaaaaaa aaaaaaaaaa ctcgtgccga attc 6644 

What is claimed is:
 1. An isolated DNA encoding smooth muscle-type myosin heavy chain SM1 isoform protein as set forth in SEQ ID NO:3.
 2. The DNA of claim 1, wherein said DNA consists of nucleotides 105 to 6020 of SEQ ID NO:
 1. 3. A vector containing the DNA of claim
 1. 4. The vector of claim 3, further comprising a promoter operably linked to the DNA.
 5. The vector of claim 3, wherein said vector is a plasmid.
 6. The vector of claim 5, wherein said plasmid is pSE-SM1-Hyg.
 7. The vector of claim 3, wherein said vector is a viral vector.
 8. The vector of claim 7, wherein said viral vector is a retrovirus vector or an adenovirus vector.
 9. A cultured host cell comprising the vector of claim
 3. 10. The host cell of claim 9, wherein said cultured host cell is a microorganism.
 11. The host cell of claim 9, wherein said cultured host cell is an animal cell.
 12. A pharmaceutical composition comprising the vector of claim 3 and a pharmaceutically acceptable carrier.
 13. A method of inhibiting vascular smooth muscle cell proliferation following angioplasty comprising administering a therapeutically effective amount of a vector comprising a DNA sequence encoding myosin heavy chain SM1 isoform polypeptide that inhibits vascular smooth muscle cell proliferation, wherein the vector is administered to a patient by using an infusion catheter so the vector is at or near the site of angioplasty.
 14. A method of treating restenosis following angioplasty comprising administering a therapeutically effective amount of a vector comprising a DNA sequence encoding myosin heavy chain SM1 isoform polypeptide that treats restenosis, wherein the vector is administered to a patient by using an infusion catheter so the vector is at or near the site of angioplasty.
 15. The method of claim 13, wherein the myosin heavy chain SM1 isoform polypeptide comprises SEQ ID NO:3.
 16. The method of claim 14, wherein the myosin heavy chain SM1 isoform polypeptide comprises SEQ ID NO:3. 